Agents capable of downregulating an MSF-A-dependent HIF-1alpha and use thereof in cancer treatment

ABSTRACT

Methods and pharmaceutical compositions for the treatment of cancer or acute ischemia are provided. Also provided are methods of identifying agents capable of preventing the formation of or dissociating the MSF-A-HIF-1alpha protein complex, and methods of determining the prognosis of an individual having cancer by identifying the presence or absence of such a protein complex.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.11/632,231 filed on Jan. 11, 2007 which is a National Phase of PCTPatent Application No. PCT/IL2005/000736 having International FilingDate of Jul. 12, 2005, which claims the benefit of priority of U.S.Provisional Patent Application No. 60/586,697 filed on Jul. 12, 2004.The contents of the above applications are all incorporated herein byreference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to agents which can prevent the formationof, or dissociate or destabilize an MSF-A-HIF1α protein complex and,more particularly, to the use of such agents in treating cancer.

Hypoxia-inducible factors (HIFs) are transcription factors involved inthe transcription activation of several genes including, angiogenicfactors (e.g., VEGF and FLT1), glucose transporters (Glut-1 and Glut-3),and glycolytic enzymes which are involved in the production of ATP inthe absence of O₂ (for overview see Semenza G L. 2003; Nat. Rev. Cancer3: 721-732; Paul S A, Simons J W, Mabjeesh N J. 2004; J. Cell Physiol.200: 20-30). HIF transcription factors are composed of two subunits,HIF-α and HIF-β. While the HIF-β is constitutively expressed, theexpression of HIF-α is regulated by the level of oxygen. Thus, in thepresence of normal oxygen tension (i.e., normoxia), HIF-1α ishydroxylated at the two critical proline residues (402 and 564 ofGenBank Accession No. NP_(—)001521) by members of the prolyl hydroxylaseprotein (PHD) family (PHD-1, -2, and -3). Hydroxylated-HIF-1α can thenbind the von Hippel-Lindau (VHL) tumor suppressor protein, whichrecruits the E3 ubiquitin-ligase complex to targeting the HIF-α proteinto proteasomal degradation. However, since oxygen is the rate-limitingco-factor of PHD enzymes, at low oxygen tension (i.e., hypoxiaconditions), the prolyl hydroxylases are unable to hydroxylate HIF-α. Asa result, no VHL interaction occurs and the E3 ubiquitin-ligase complexis unable to target HIF-1α to proteasomal degradation, resulting instabilization of HIF. Stabilized HIF-1α can then form a heterodimer withthe HIF-1β, which interacts with the basic helix-loop-helix domain ofthe hypoxia response element (HRE) in target genes.

In addition, hydroxylation of an asparagine residue in the C-terminaltransactivation domain (TAD) of HIF-α (at position 803) by the factorinhibiting HIF-1 (FIH-1) negatively regulates transcriptional activityof HIF by preventing its interaction with p300 and CBP transactivators.

Elevated levels of HIF-1α protein are found in the majority of solidtumors and cancer metastases in the areas of profound hypoxia (QuinteroM, Mackenzie N, Brennan P A. 2004; Eur. J. Surg. Oncol. 30: 465-8). Inaddition, a number of oncogenes such as AKT, Src, and oncogenic Ras werefound to induce HIF expression (Li J, et al., 2004; Cancer Res. 64:94-101). Moreover, p53 and Hsp90 were found to positively and negativelyregulate HIF-1α degradation, respectively, i.e., while under normoxiaP53 promotes HIF-1α degradation (Choi K S et al., 2003; J. Biochem. Mol.Biol. 36: 120-7), Hsp90 has a protective role in VHL-independentdegradation of HIF-1α (Isaacs J S et al., J. Biol. Chem. 2004; 279:16128-35). In addition, in many cases, the major reason for the failureof cancer therapy is the resistance of hypoxic cancer cells to bothchemotherapy and radiation (Escuin D et al., 2004; Cancer Biol Ther.3(7). Epub ahead of print). Thus, HIF-1α has been recognized as apossible target for anti cancer therapy (Welsh S J and Powis G. 2003;Curr Cancer Drug Targets. 3(6): 391-405; Macpherson G R and Figg W D,2004; Cancer Biol. Ther. 3(6). Epub ahead of print).

Several agents capable of downregulating HIF-1 have been identified aspotential anti-cancer agents including FK228, a histone deacetylase(HDAC) inhibitor (Mie Lee Y et al., 2003. Biochem. Biophys. Res. Commun300: 241-6), PX-478, a small-molecule HIF-1 inhibitor, (Macpherson G R,Figg W D. 2004. Cancer Biol. Ther. 3(6) Epub ahead of print) andBisphenol A, an environmental endocrine-disrupting chemical (Kubo T etal., 2004; Biochem. Biophys. Res. Commun 318(4): 1006-11). However,although desired, the mechanisms leading to up- or down-regulation ofHIFs in cancerous tumors are not yet clear, thus, limiting the use ofHIF-1 inhibitors/suppressors as anti cancer agents.

While reducing the present invention to practice, the present inventorhas uncovered that MSF-A, a myeloid/lymphoid leukemia septin-like fusionprotein A, regulates HIF-1α activity and thus contributing to cancerprogression.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided amethod of treating cancer and/or inhibiting a growth of a canceroustumor and/or metastases in an individual comprising providing to theindividual an agent capable of downregulating an MSF-A-dependent HIF-1αactivity in cells of the individual thereby treating the cancer and/orinhibiting the growth of the cancerous tumor and/or the metastases inthe individual.

According to another aspect of the present invention there is providedan antibody or antibody fragment capable of specifically binding to anMSF-A polypeptide.

According to yet another aspect of the present invention there isprovided a method of treating acute ischemia in cells of an individualcomprising providing to the individual an agent capable of increasing anMSF-A-dependent HIF-1α activity in cells of the individual to therebytreat the acute ischemia.

According to still another aspect of the present invention there isprovided a method of identifying putative anti cancer agents, the methodcomprising identifying agents being capable of preventing the formationof and/or dissociating an MSF-A-HIF-1α protein complex, therebyidentifying the putative anti cancer agents.

According to an additional aspect of the present invention there isprovided a method of determining if a molecule is capable of preventingthe formation of and/or dissociating an MSF-A-HIF-1α protein complex,comprising incubating the MSF-A-HIF-1α protein complex or cellsharboring the MSF-A-HIF-1α protein complex with the molecule anddetermining the presence or absence of the MSF-A-HIF-1α protein complexfollowing the incubating, wherein the absence of the MSF-A-HIF-1αprotein complex is indicative of the capacity of the molecule to preventthe formation of and/or dissociate the MSF-A-HIF-1α protein complex.

According to yet an additional aspect of the present invention there isprovided a method of determining the prognosis of an individual havingcancer, comprising determining the presence or absence of anMSF-A-HIF-1α protein complex in cancerous cells derived from theindividual, wherein the presence of the MSF-A-HIF-1α protein complex isindicative of poor prognosis of the individual.

According to further features in preferred embodiments of the inventiondescribed below, downregulating the MSF-A-dependent HIF-1α activity iseffected by preventing a formation of an MSF-A-HIF-1α complex and/ordissociating the MSF-A-HIF-1α complex.

According to still further features in the described preferredembodiments the agent capable of preventing formation of theMSF-A-HIF-1α complex is capable of downregulating and/or preventing anassociation between MSF-A and HIF-1α.

According to still further features in the described preferredembodiments the agent capable of preventing the formation of and/ordissociating the MSF-A-HIF-1α protein complex is selected from the groupconsisting of an MSF-A antisense oligonucleotide, an MSF-A siRNA, anMSF-A DNAzyme, an MSF-A Ribozyme, an MSF-A antibody or antibodyfragment, a non-functional MSF-A polypeptide, an MSF-A derived peptideor peptide analog, a non-functional HIF-1α polypeptide and an HIF-1αderived peptide or peptide analog.

According to still further features in the described preferredembodiments the MSF-A antibody or antibody fragment is capable ofspecifically binding to the polypeptide set forth by SEQ ID NO:3.

According to still further features in the described preferredembodiments the non-functional MSF-A polypeptide is set forth by SEQ IDNO:10.

According to still further features in the described preferredembodiments the MSF-A derived peptide or peptide analog includes theamino acid sequence set forth in SEQ ID NOs:2463-4193 or 4213.

According to still further features in the described preferredembodiments the HIF-1α derived peptide or peptide analog includes theamino acid sequence set forth in SEQ ID NOs:12-2462.

According to still further features in the described preferredembodiments the cancer and/or the cancerous tumor is selected from thegroup consisting of prostate cancer, breast cancer, chemotherapy-inducedMLL, stomach cancer, cervical cancer, endometrial cancer, and ovariancancer.

According to still further features in the described preferredembodiments the antibody is capable of preventing the formation ofand/or dissociating an MSF-A-HIF-1α protein complex.

According to still further features in the described preferredembodiments the acute ischemia is a result of stroke and/or acutemyocardium infraction. According to still further features in thedescribed preferred embodiments increasing the MSF-A-dependent HIF-1αactivity is effected by upregulating formation of an MSF-A-HIF-1αprotein complex and/or stabilizing the MSF-A-HIF-1α protein complex.

According to still further features in the described preferredembodiments the agent capable of upregulating the MSF-A-HIF-1α proteincomplex is capable of increasing the association between MSF-A andHIF-1α.

According to still further features in the described preferredembodiments the agent capable of upregulating and/or stabilizing theMSF-A-HIF-1α protein complex is selected from the group consisting of anexogenous polynucleotide encoding at least a functional portion ofMSF-A, an exogenous polynucleotide encoding at least a functionalportion of HIF-1α, an exogenous polypeptide including at least afunctional portion of MSF-A, an exogenous polypeptide including at leasta functional portion of HIF-1α, a polypeptide capable of stabilizing theMSF-A-HIF1α protein complex.

According to still further features in the described preferredembodiments the agents are selected from the group consisting ofchemicals, antibodies, aptamers, peptides, and peptide analogs.

According to still further features in the described preferredembodiments the peptide or peptide analog is derived from MSF-A orHIF-1α. According to still further features in the described preferredembodiments the peptide or peptide analog includes the amino acidsequence set forth in SEQ ID NOs: 2463-4193, 4213 or 12-2462.

According to still further features in the described preferredembodiments incubating is effected for a time period selected from therange of 1-48 hours.

According to still further features in the described preferredembodiments the presence or the absence of the MSF-A-HIF-1α proteincomplex is effected using anti-MSF-A and/or anti-HIF-1α antibody.

According to still further features in the described preferredembodiments determining the presence or the absence of the MSF-A-HIF-1αprotein complex is effected by sequentially and/or simultaneouslyexposing the MSF-A-HIF-1α protein complex or the cells harboring theMSF-A-HIF-1α protein complex to the anti-MSF-A and the anti-HIF-1αantibodies.

According to still further features in the described preferredembodiments determining the presence or the absence of the MSF-A-HIF-1αprotein complex is effected using an immunological detection method.

According to still further features in the described preferredembodiments the immunological detection method utilizes an anti-MSF-Aand/or an anti-HIF-1α antibody or antibody fragment.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing methods of treating canceror acute ischemia.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIG. 1 is an autoradiogram illustrating HIF-1α-immunoprecipitation fromwhole cell lysates of ³⁵S-metabolically labeled PC-3 cells.

FIG. 2 is a Western Blot analysis illustrating the expression ofrecombinant MSF-A protein in HEK 293 transfected cells. HEK 293 cellswere transiently transfected with the p3xFlag-MSF-A vector and theexpression of the recombinant MSF-A protein was detected at theindicated time points using an anti-Flag antibody (Sigma-Aldrich Corp.,St Louis, Mo., USA).

FIGS. 3 a-f are autoradiograms illustrating HIF-1α immunoblotting ofFLAG immunoprecipitates (IP) (FIG. 3 b) or whole cell extracts (WCE)(FIG. 3 a), FLAG immunoblotting of HIF-1α IP (FIG. 3 d) or WCE (FIG. 3c), and HIF-1β immunoblotting of FLAG IP (FIG. 30 or WCE (FIG. 3 e). HEK293 cells were transiently co-transfected with two of the followingexpression vectors: p3xFlag-cmv-25 (EV), pcdna3.1-HIF-1α (HIF-1α) orp3xFlag-MSF-A (MSF-A), and two days following transfection undernormoxic conditions WCE or IP were subjected to immunoblot (IB)analysis. Lane 1-EV and HIF-1α; lane 2-MSF-A and HIF-1α.

FIGS. 4 a-b are autoradiograms depicting HIF-1α (FIG. 4 a) or Flag (FIG.4 b) immunoblotting of HIF-1α immunoprecipitates. HEK 293 cells wereco-transfected with the p3xFlag-HIF-1α and p3xFlag-MSF-A vectors and 24hours following transfection the cell were subjected to either normoxia(lanes 1-4) or hypoxia (lanes 5-8) for another 24 hours. Lanes 1 and5=WCE, lanes 2 and 6=IP sup, lanes 3 and 7=HIF-1α IP without an antibody(negative control), lanes 4 and 8=HIF-1α IP; WCE=whole cell extracts; IPsup=immunoprecipitation supernatant; Ab=antibody.

FIGS. 5 a-c are autoradiograms illustrating MSF-A association to theHIF-1α complex. HEK 293 cells were co-transfected with two of the threeexpression vectors as in FIGS. 3 a-f, and two days followingtransfection the cells were subjected to IP analysis using anti-HIF-1α(FIGS. 5 a-b) or anti-Flag (FIG. 5 c) antibodies, followed by IB usingthe anti-Flag (FIG. 5 a) or anti-p300 (FIGS. 5 b-c; Santa CruzBiotechnology Inc., Santa Cruz, Calif.) antibodies. Lanes 1 and 3-EV andHIF-1α; lanes 2 and 4-MSF-A and HIF-1α.

FIG. 6 is a graph illustrating reporter gene activity under normoxia orhypoxia. PC-3 cells were co-transfected with a plasmid expressing theluciferase gene under the control of hypoxia response element (HRE) andwith either the p3xFlag-cmv-25 (EV) or the p3xFlag-MSF-A vector.Twenty-four hours following transfection, cells were left under normoxiaor were subjected to hypoxia for overnight, following which theluciferase luminescence assay was employed. Results are expressed asaverage of triplicates. P value<0.05.

FIG. 7 is a graph depicting luciferase activity (a reporter of HIF-1αtranscriptional activation) as a function of the presence or absence ofthe N-terminus of the MSF-A protein. HEK 293 cells were co-transfectedwith 0.1 μg HRE-dependent luciferase reporter 1pBI-GL V6L (Post, D. E.,and Van Meir, E. G. 2001. Generation of bidirectionalhypoxia/HIF-responsive expression vectors to target gene expression tohypoxic cells. Gene Ther 8, 1801-1807., Mabjeesh, N. J., et al., 2002,Geldanamycin induces degradation of hypoxia-inducible factor 1alphaprotein via the proteasome pathway in prostate cancer cells. Cancer Res62, 2478-2482) and the wild type (WT) MSF-A (p3xFlag-MSF-A), ΔN-MSF-A(ΔN) (p3xFlag-ΔN-MSF-A) or empty vector (EV) (p3xFlag-cmv-25) constructsas follows: Experiment (Exp.) No. 1=1 μg EV; Exp. No. 2=0.5 μg EV and0.5 μg WT; Exp. No. 3—1 μg WT; Exp. No. 4=0.5 μg EV and 0.5 μg ΔN; Exp.No. 5=1 μg ΔN. After 24 hours of transfection, cells were subjected tonormoxia or hypoxia for overnight and then analyzed for luciferaseluminescence assay. Luciferase activity is presented as relative unitsper μg protein in each experiment. Columns represent means; barsrepresent SD; n=3; *, p<0.05 compared to hypoxia of EV. Note thesignificant decrease of luciferase activity in cells grown under hypoxiaand transfected with the N-terminus truncated form (ΔN) of MSF-A ascompared with cells transfected with the wild-type form of MSF-A. Alsonote that under normoxia, when the luciferase activity is hardlydetected, no significant difference is obtained.

FIG. 8 is a graph depicting luciferase activity (a reporter of HIF-1αtranscriptional activation) as a function of the presence or absence ofthe GTP binding site of MSF-A (ΔG). HEK 293 cells were cotransfectedwith HRE-dependent luciferase reporter and the expression vectorencoding wild-type MSF-A (WT) (p3xFlag-MSF-A), the deleted GTP bindingsite form of MSF-A (ΔG) (p3xFlag-ΔG-MSF-A) or empty vector (EV)(p3xFlag-cmv-25) and then were exposed to hypoxia conditions asdescribed in the description of FIG. 4, hereinabove. Luciferase activityis presented as relative units per μg protein in each experiment.Columns represent means; bars represent SD; n=3. Note thenon-significant effect of the ΔG-MSF-A on HIF-1α activity.

FIGS. 9 a-c are Western blot analyses of FLAG (MSF-A; FIGS. 9 a and b)and HIF-1α (FIG. 9 c) depicting the interaction between HIF-1α and theWT or deleted forms of MSF-A. HEK 293 cells were transientlyco-transfected with expression vector encoding Flag-MSF-A (WT;p3xFlag-MSF-A), Flag-ΔG (ΔG; p3xFlag-ΔG-MSF-A), Flag-ΔN (ΔN;p3xFlag-ΔN-MSF-A) or empty vector (EV; p3xFlag-cmv-25). After 48 hours,the cells were lysed, subjected for immunoprecipitation (IP) usingHIF-1α antibody and then immunoblotted (IB) with HIF-1α or Flagantibodies. FIG. 9 a—whole cell extracts (WCE) subjected to IB with theFlag antibody (MSF-A); FIG. 9 b—IP prepared by the HIF-1α antibody weresubjected to IB with the Flag antibody (MSF-A); FIG. 9 c—IP prepared bythe HIF-1α antibody were subjected to IB with the HIF-1α antibody.

FIGS. 10 a-b depict MSF-A expression (FIG. 10 a) and activation ofHIF-1α (FIG. 10 b) in MSF-A stably transfected PC-3 cells. FIG. 10 a isan immunoblot of PC-3 cells stably transfected with the MSF-A vector(p3xFLAG-MSF-A; clones numbers 8, 9, 10, 11, 12, 13, 15, 16, 17, 20, 22,23, 24, 25, 28, 29, 30) or the EV (p3xFlag-cmv-25) using the anti-Flagantibody. Neomycin-resistant clones were grown under normoxicconditions, harvested and analyzed for MSF-A expression byimmunoblotting with Flag antibody. Note the high expression level ofMSF-A in stably transfected clones numbers (#) 7, 8, 9, 10, 11, 12, 13,15, 16, and 17. FIG. 10 b is a graph depicting luciferase activity invarious stably MSF-A transfected cells. Parental PC-3 cells and selectedstably transfected clones [transfected with the EV or MSF-A (clonenumbers 7, 11 and 25)] were transiently transfected with 1 μg of theHRE-dependent luciferase reporter (pBI-GL V6L) for HIF-1 transcriptionalactivity. Luciferase activity is presented in units per mg protein ineach transfected cells. Columns=means; bars=SD; n=3; *, p<0.05 comparedto hypoxia of EV.

FIG. 11 is a graph depicting enhancement of luciferase activity in cellsstably transfected with MSF-A. Pooled clones of PC-3 cells stablytransfected with the MSF-A expression vector (MSF-A) or the empty vector(Neo) were subjected to reporter luciferase assay under normoxia orhypoxia conditions. Note the significant difference in luciferaseactivity under hypoxia in cells stably expressing the MSF-A protein ascompared with cells transfected with the empty vector.

FIGS. 12 a-f are RT-PCR analyses depicting HIF-1α mediated activation ofvarious genes in cells transfected with the expression vector alone(Neo) or the MSF-A expression vector (MSF-A). Total RNA was isolatedfrom PC-3-Neo and PC-3-MSF-A cells grown under normoxic (lanes 1 and 2)and hypoxic (lanes 3 and 4) conditions. Semi-quantitative RT-PCRanalysis was performed using VEGF, Glut-1, ET-1, CA-IX, HIF-1α andβ-actin primers (SEQ ID NOs:4200-4211 as described under GeneralMaterials and Experimental Methods). Lane 1—Neo, lane 2—MSF-A, lane3—Neo, lane 4—MSF-A, lane 5—water (negative control). Note thesignificant increased expression of VEGF in PC-3-MSF-A cells grown underhypoxia (lane 4 in FIG. 12 a) as compared with PC-3-MSF-A cells grownunder normoxia (lanes 2 in FIG. 12 a) as well as compared with PC-3-Neocells grown under either hypoxia (lane 3 in FIG. 12 a) or normoxia (lane1 in FIG. 12 a).

FIG. 13 is a graph depicting the effect of MSF-A over-expression on cellproliferation. PC-3-Neo and PC-3-MSF-A cells were grown under normoxicand hypoxic conditions for the indicated time and then analyzed forproliferation using XTT assay. Proliferation was expressed as increasein % of the initial O.D. measured on the next day of seeding which wasconsidered 100%. Growth media were not changed until the end of theexperiment.

FIGS. 14 a-g are microscopic photographs (FIGS. 14 a-f) and a graphdepicting the effect of MSF-A over-expression on colony formation andgrowth (FIG. 14 g). PC-3-Neo and PC-3-MSF-A cells were grown for 4 weeksin soft agar under normoxic conditions. Colonies were observed andcounted. FIGS. 14 a, c, and e—representative colonies from each plateseeded with PC-3-Neo cells; FIGS. 14 b, d, and f—representative coloniesfrom each plate seeded with PC-3-MSF-A cells; FIG. 14 g-quantitativeanalysis of colony number from each cell type. Columns=means; bars=SD;n=3.

FIGS. 15 a-b are Western blot analyses depicting HIF-1α (FIG. 15 a) andactin (FIG. 15 b) expression level in PC-3-Neo and PC-3-MSF-A cellsgrown under normoxia (N) or subjected to hypoxia (H). PC-3-Neo cells(lanes 9-16) or PC-3-MSF-A cells (lanes 1-8) were grown under normoxia(lanes 1-2,5-6, 9-10, 13-14) or hypoxia (lanes 2-4,7-8, 11-12, 15-16)for 4 hours (lanes 1-4 and 9-12) or 8 hours (lanes 5-6 and 13-16) asindicated. Cytosolic (CE; lanes 1, 3, 5, 7, 9, 11, 13, and 15) ornuclear (NE; lanes 2, 4, 6, 8, 10, 12, 14, and 16) extracts wereprepared, analyzed by SDS-PAGE, and immunoblotted with antibodies toHIF-1α and actin.

FIGS. 16 a-c are Western blot analyses depicting the effect ofcycloheximide (CHX) on the expression level of HIF-1α (FIG. 16 a) andα-tubulin (FIG. 16 b) in PC-3-Neo or PC-3-MSF-A cells grown undernormoxia. CHX was added to PC-3-Neo (lanes 1-5) or PC-3-MSF-A (lanes6-10) at a final concentration of 10 μg/ml, for 0 (lanes 1 and 6), 5(lanes 2 and 7), 15 (lanes 3 and 8), 30 (lanes 4 and 9) and 45 (lanes 5and 10) minutes. Whole cell extracts were prepared and resolved bySDS-PAGE and Western blotting was performed with antibodies againstHIF-1α (FIG. 16 a) or α-tubulin (FIG. 16 b). FIG. 16 c is a graphdepicting quantitation of the results obtained in FIGS. 16 a and bexpressed as the ratio of HIF-1α expression level normalized to that ofα-tubulin. Note, the relatively slow degradation of HIF-1α in cellsover-expressing the MSF-A protein, with a degradation half-life of 40minutes as compared to the degradation half-life of less than 20 minutesin PC-3-Neo cells (FIG. 16 c).

FIGS. 17 a-b depict pulse-chase analysis of HIF-1α. PC-3-Neo andPC-3-MSF-A cells were labeled with ³⁵S-methionine and pulse-chased incomplete medium containing for the indicated time in hours (h). FIG. 17a-equal amounts of protein from each cell lysate were subjected toimmunoprecipitation with anti-HIF-1α antibody, resolved by SDS-PAGE andsubjected to autoradiography. FIG. 17 b-quantification of theautoradiographic HIF-1α signal by densitometry. Note, the relativelyslower degradation of HIF-1α in cells over-expressing the MSF-A protein,with a degradation half-life of 45 minutes as compared to thedegradation half-life of 25 minutes in PC-3-Neo cells.

FIGS. 18 a-b depict stabilization of HIF-1α in PC-3-MSF-A stablytransfected cells. Whole cell lysates from single clones of stablytransfected PC-3 cells (expressing different amounts of Flag-MSF-Aprotein) were grown under normoxia (lanes 1-6) or hypoxia (lanes 7-12)were analyzed on SDS-PAGE and immunoblotted with HIF-1α (FIG. 18 a) andFlag (FIG. 18 b) antibodies. Note the significant reduction (underhypoxia conditions) in the ubiquitinated species of HIF-1α in MSF-Astably transfected cells (clones 1-5; lanes 8-10 and 12 in FIG. 18 a) ascompared to cells transfected with the expression vector alone (Neo;lane 7 in FIG. 18 a). Also note the correlation between the level ofMSF-A expression and the inverse effect on HIF-1α ubiquitinated species(compare e.g., lane 11 to 8 in FIGS. 18 a and b).

FIGS. 19 a-b are Western blot analyses depicting the effect ofproteasome inhibition on HIF-1α (FIG. 19 a) or actin (FIG. 19 b)expression levels in PC-3-MSF-A or PC-3-Neo cells. PC-3-Neo (lanes 1-3)and PC-3-MSF-A (lanes 4-6) cells were treated for 4 hours with either0.1 DMSO (lanes 1 and 4) or with 5 (lanes 2 and 5) and 20 (lanes 3 and6) μM MG-132. Whole cell lysates were prepared and equal amounts ofprotein from each cell lysate were resolved by SDS-PAGE, transferred andimmunoblotted with antibodies against HIF-1α (FIG. 19 a) and actin (FIG.19 b). Ub-HIF-1α points to ubiquitinated HIF-1α protein species.

FIGS. 20 a-b are Western blot analyses of HIF-1α immunoprecipitatesdepicting the expression level of HIF-1α (FIG. 20 a) and ubiquitin (FIG.20 b) in PC-3-Neo and PC-3-MSF-A cells. PC-3-Neo (lanes 1-2) andPC-3-MSF-A (lanes 3-4) cells were treated for 4 hours with either 0.1DMSO (lanes 1 and 3) or with 10 μM MG-132 (lanes 2 and 4). Whole celllysates were prepared and subjected to immunoprecipitation (IP) withHIF-1α antibody Immunoprecipitates were resolved on SDS-PAGE andimmunoblotted (IB) with HIF-1α (FIG. 20 a) and ubiquitin (FIG. 20 b)antibodies. Ub-HIF-1α points to ubiquitinated HIF-1α protein species.

FIGS. 21 a-c are Western Blot analyses illustrating the specificity ofthe anti-MSF-A immune serum. PC-3 cells were transfected with either theexpression vector (EV) or the p3xFlag-MSF-A vector (MSF-A) and weresubjected to Western Blot analyses (IB) using the anti-Flag antibody(FIG. 21 a), preimmune serum (FIG. 21 b) or serum after immunizationwith a peptide corresponding to the N-terminal of MSF-A protein (FIG. 21c). Note the presence of two MSF-A positive bands in PC-3 cellstransfected with the p3xFlag-MSF-A vector, corresponding to the Flag—andendogenous MSF-A proteins (FIG. 21 c).

FIGS. 22 a-d FLAG (FIGS. 22 a-b) or sera-raised MSF-A (FIGS. 22 c-d)immunoblot analyses of FLAG IP (FIGS. 22 a and c) or whole cell extracts(FIGS. 22 b and d). HEK 293 were transfected with expression vectorencoding Flag-MSF-A (lane 2) or empty vector (EV, lane 1) and whole cellextracts were prepared. Lysates were subjected to immunoprecipitation(IP) with Flag antibody Immunoprecipitates were resolved on SDS-PAGE andanalyzed by immunoblotting (IB) with Flag antibody or sera raisedagainst the N-terminus of MSF-A. FIG. 22 a—IP with FLAG and IB withFLAG; FIG. 22 b—no IP and IB with FLAG; FIG. 22 c—IP with FLAG and IBwith sera-raised MSF-A; FIG. 22 d—no IP and IB with the sera-raisedMSF-A.

FIGS. 23 a-c are Western blot analyses depicting the expression ofHIF-1α (FIG. 23 a) and MSF-A (FIG. 23 b) in the nuclear rather than thecytosolic cell fraction. PC-3 or CL-1 cells were grown for 24 hoursunder either normoxia or hypoxia following which the expression level ofHIF-1α (FIG. 23 a), MSF-A (FIG. 23 b) or α-tubulin (FIG. 23 c) wasdetected using Western blot analysis. CE=cytosolic extract; NE=nuclearextract; N=normoxia; H=hypoxia. Note that while MSF-A and HIF-1αlocalize at the cell nuclear fraction, the α-tubulin protein localizesat both the nuclear and cytoplasm fractions.

FIGS. 24 a-d illustrate MSF-A immunofluorescence staining in PC-3 cellsusing MSF-A preimmune (FIGS. 24 c-d) or immune sera (FIGS. 24 e-f) undernormoxia (FIGS. 24 c and e) or hypoxia (FIGS. 24 d and f).

FIGS. 25 a-f illustrate HIF-1α and MSF-A co-localization in PC-3 cellsusing anti-HIF-1α (FIGS. 25 a-b), anti-MSF-A (FIGS. 25 c-d) or bothantibodies (FIGS. 25 e-f) under normoxia (FIGS. 25 a, c, e) or hypoxia(FIGS. 25 b, d, f).

FIGS. 26 a-c are Western Blot analyses of whole cell extracts (WCE) orHIF-1α-IP using anti-HIF-1α (FIG. 26 a) or anti-MSF-A (FIGS. 26 b-c)antibodies. PC-3 (FIGS. 26 a-b) or CL-1 (FIG. 26 c) cells were grownunder normoxia or hypoxia and whole cell extracts orHIF-1α-immunoprecipitates were subjected to Western Blot analyses. Lanes1 and 3—normoxia; lanes 2 and 4—hypoxia.

FIG. 27 is a schematic presentation depicting MSF-A involvement in theregulation of HIF.

FIG. 28 is a graph depicting the effect of MSF-A over-expression ontumor volume. A prostate cancer xenograft model was established usingPC-3-Neo and PC-3-MSF-A cells (3×10⁶) which were implantedsubcutaneously into the right hind of nude mice. PBS was used as anegative control Animals were monitored for tumor volume measurements.Tumor volume measurements were calculated using the formulawidth²×length×0.52. Mean±SEM (n=5) of representative experiments isshown. Note the significant increase in tumor volume in xenograftsinjected with cells over-expressing the MSF-A protein (PC-3-MSF-A).

FIG. 29 is a graph depicting the effect of MSF-A over-expression ontumor weight. A prostate cancer xenograft model was established asdescribed in FIG. 28. Mice were sacrificed after 6 weeks and tumors wereprocessed for tumor weight measurements. Tumors were weighed immediatelyafter dissection. Columns, means; bars, SEM; n=5; *, p<0.05. Note thesignificant increase in tumor weight in xenografts injected with cellsover-expressing the MSF-A protein (PC-3-MSF-A).

FIGS. 30 a-h are photomicrographs (FIGS. 30 a-f) and graphs (FIGS. 30g-h) depicting MSF-A over-expression in tumors and angiogenesis in aprostate cancer xenograft model. Sections from both PC-3-Neo (FIGS. 30a, c, and e) and PC-3-MSF-A (FIGS. 30 b, d, and f) tumors were subjectedfor Hematoxylin-eosin (H& E; FIGS. 30 a-b) and immunostaining with Ki67(FIGS. 30 c and d) and CD34 (FIGS. 30 e and f). FIG. 30 g-Ki67 staining(%) was quantified by dividing the number of positive nuclei by thenumber of total nuclei in 40× magnification field multiplied by 100, of5 paraffin-embedded tumor sections from each animal per group. Columns,average of the means of Ki67 staining from each animal; bars, SEM; n=5;*, p<0.05. FIG. 30 h-Microvessel density (MVD) was determined bycounting the capillaries positive for CD34 staining in 4× magnificationfield per total section area excluding necrotic areas in 5paraffin-embedded tumor sections from each animal per group. Columns,average of the means of MVD from each animal; bars, SEM; n=5; *, p<0.05.

FIGS. 31 a-f are RT-PCR analyses of MSF-A (FIG. 31 a), VEGF (FIG. 31 b),CA-IX (FIG. 31 c), Glut-1 (FIG. 31 d), ET-1 (FIGS. 31 e) and β-actin(FIG. 31 f) of RNA isolated from either PC-3-Neo (lane 1) or PC-3-MSF-A(lane 2)-derived tumors. Lane 3=water (negative control).

FIGS. 32 a-d depict MSF-A mRNA expression in human tumors. FIG. 32 a isa graph depicting normalized MSF-A expression levels in various tumors.Human Matched Tumor/Normal Expression Array (containing 68 pairs oftumor/normal different tissues) was hybridized with probe to MSF-A (SEQID NO:4214, the probe also reacts with other SEPT9 transcripts) and toβ-actin (Ambion). Autoradiograms were analyzed, and the expression ratioof each tumor/normal was depicted in the graph. Numbers in parenthesispoint to the number of pairs of each tumor type. FIGS. 32 b-c are RT-PCRanalyses of MSF-A (FIGS. 32 b) and β-actin (FIG. 32 c) in various normaland cancerous tissues or cells. Total RNA was isolated from twodifferent normal prostate tissues [NP, NP#1 (lane 1), NP#2 (lane 2)],prostate cancer cell lines [PC-3 (lane 3), CL-1 (lane 4), LNCaP (lane5)1 and prostate cancer xenografts [LuCaP 35 (lane 6), LAP-C9 (lane 7),WISH-PC-14 (lane 8), WM 2C3 (lane 9)], and was analyzed by RT-PCR usingprimers specific to MSF-A (SEQ ID NOs:4 and 4199) and β-actin (SEQ IDNOs:4202-4205). FIG. 32 d is a graph depicting normalization of theRT-PCR analyses shown in FIGS. 32 b and c. Shown are the averagedensitometric quantification of MSF-A/actin expression in the normalprostate tissues (NP), the prostate cancer cell lines and xenografts.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of agents capable of preventing the formationof, and/or dissociating an MSF-A-HIF-1α protein complex or of agentscapable of stabilizing the MSF-A-HIF-1α protein complex which can beused to treat cancer or acute ischemia, respectively. Specifically, thepresent invention can be used to treat individuals having cancer usingagents capable of downregulating the MSF-A and/or HIF-1α proteins.

The principles and operation of the methods of treating cancer or acuteischemia according to the present invention may be better understoodwith reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

Over-expression of HIF-1α is found in the majority of solid tumors andcancer metastases in the areas of profound hypoxia [Quintero, 2004(Supra)]. In addition, in many cases, the major reason for the failureof anti cancer therapy is the resistance of hypoxic cancer cells to bothchemotherapy and radiation [Escuin, 2004 (Supra)]. Thus, HIF-1α has beenrecognized as a possible target for anti cancer therapy [Welsh, 2003(Supra)].

Several agents capable of downregulating HIF-1 have been identified aspotential anti-cancer agents including FK228, a histone deacetylase(HDAC) inhibitor [Mie Lee, 2003 (Supra)], PX-478, a small-molecule HIF-1inhibitor, [Macpherson, 2004 (Supra)] and Bisphenol A, an environmentalendocrine-disrupting chemical [Kubo, 2004 (Supra)]. However, themechanisms leading to up- or down-regulation of HIFs in cancerous tumorsare not yet clear, thus, limiting the use of HIF-1inhibitors/suppressors as anti cancer agents.

While reducing the present invention to practice, the present inventorhas uncovered that MSF-A, a myeloid/lymphoid leukemia septin-like fusionprotein A, associates with HIF-1α both in vitro and in vivo (Example 1,FIGS. 1-5) and that MSF-A over-expression upregulates HIF-1αtranscriptional activity (Example 2, FIG. 6). When in a complex withHIF-1α, MSF-A prevents proteasomal degradation of HIF-1α (Example 5,FIGS. 15-20). Moreover, as is further shown in FIGS. 28-32 and isdescribed in Examples 8 and 9 of the Examples section which follows,MSF-A is over-expressed in various tumors and is capable of inducingtumor growth, angiogenesis and proliferation in vivo. These findingshave led the present inventor to design agents which can be used totreat cancer by downregulating MSF-A dependent HIF-1α activity.

As described in Example 3 of the Examples section which follows,transfection of cells with the p3xFlag-ΔN-MSF-A expression vectorencoding an N-terminal truncated form of the MSF-A protein resulted ininhibition of HIF-1α activation below the level observed in cellstransfected with the empty vector (i.e., wild type, FIG. 7). Inaddition, transfection of cells with the p3xFlag-ΔG-MSF (lacking the GTPbinding domain of MSF-A) resulted in lack of activation of HIF-1αtranscriptional activity (levels of activation were similar to thoseobserved with the empty vector, FIG. 8). On the other hand, as is shownin FIGS. 9 a-c, both of these mutants (i.e., ΔG-MSF and ΔN-MSF) wereco-immunoprecipitated with the HIF-1α protein demonstrating theirability to interfere with HIF-1α activity via the formation of theprotein complex between HIF-1α and MSF-A.

Thus, according to one aspect of the present invention there is provideda method of treating cancer and/or inhibiting a growth of a canceroustumor and/or metastases in an individual. The method is effected byproviding to the individual an agent capable of downregulating anMSF-A-dependent HIF-1α activity in cells of the individual therebytreating the cancer and/or inhibiting the growth of the cancerous tumorand/or the metastases in the individual.

As used herein, the term “individual” includes mammals, preferably humanbeings at any age. Preferably, this term encompasses individuals whichhave been diagnosed with cancer, i.e., they have cancerous cells, acancerous tumor and/or cancer metastases.

The phrase “treating” refers to inhibiting or arresting the developmentof a disease, and/or causing the reduction, remission, or regression ofa disease, in an individual suffering from, or diagnosed with, thedisease. Those of skill in the art will be aware of variousmethodologies and assays which can be used to assess the development ofa disease, and similarly, various methodologies and assays which can beused to assess the reduction, remission or regression of a disease.

The terms “cancer” and/or “cancerous tumor” as used herein encompasssolid and non-solid tumors such as prostate cancer, breast cancer,chemotherapy-induced MLL, stomach cancer, cervical cancer, endometrialcancer, ovarian cancer and the like.

As used herein the term “HIF-1α” refers to the hypoxia-inducible factor1, alpha subunit isoform 1 (SEQ ID NO:11; GenBank Accession No.AAP88778), which is a member of the Per-ARNT-Sim (PAS) superfamily 1 andan aryl hydrocarbon receptor nuclear translocator (ARNT) interactingprotein.

The phrase “MSF-A-dependent HIF-1α activity” as used herein, refers toHIF-1α protein activity (e.g., transcriptional activation of genes suchas VEGF) which is dependent on the direct or indirect interaction withMSF-A, and/or on the activation, stabilization and/or prevention ofdegradation which is mediated by MSF-A.

As used herein the term “MSF-A: refers to the myeloid/lymphoid leukemiaseptin-like fusion protein A (MSF-A, GenBank Accession No. AAF23374, SEQID NO:3).

Downregulating an MSF-A-dependent HIF-1α activity can be effected byvarious approaches including, for example, directly or indirectlyinterfering with MSF-A dependent HIF-1α protein stabilization, promotingMSF-A dependent HIF-1α protein degradation and/or preventing theformation of MSF-A-HIF-1α complex or dissociating a pre-existingMSF-A-HIF-1α complex.

It will be appreciated that several approaches can be used to preventthe formation of and/or dissociate the MSF-A-HIF-1α protein complex incells. These include downregulation of the expression level and/oractivity of any of the proteins in the protein complex (i.e., MSF-Aand/or HIF-1α) and thus preventing MSF-A-HIF-1α complex formation,interference with the protein complex or destabilization thereof.

Downregulation of MSF-A and/or HIF-1α can be effected on the genomicand/or the transcript level using a variety of molecules which interferewith transcription and/or translation (e.g., antisense, siRNA, Ribozyme,DNAzyme), or on the protein level using e.g., antagonists, antibodies,enzymes that cleave the polypeptide and the like. Preferably, agentswhich are capable of preventing the association between HIF-1α and MSF-Aare suitable for use along with the present invention.

Following is a list of agents capable of downregulating expression leveland/or activity of MSF-A or HIF-1α and as such are suitable for use withthe method of the present invention.

One example of an agent capable of downregulating MSF-A-dependent HIF-1αactivity, preventing the formation of an MSF-A-HIF-1α complex ordestabilizing an already formed complex is an antibody or antibodyfragment capable of specifically binding MSF-A or HIF-1α. Such anantibody can be a neutralizing antibody which binds an epitope on MSF ofHIF-1α and thus inhibits MSF-A-dependent HIF-1α activity. Preferably,the antibody specifically binds at least one epitope of a MSF-A orHIF-1α. Non-limiting examples of such epitopes are set forth by SEQ IDNO:4213 or 4198. Measures are taken though, to select an epitope whichwill be specifically recognized by the neutralizing antibody. Forexample, a suitable antibody which can be used along with the presentinvention is an anti-MSF-A antibody or antibody fragment as described inExample 6 of the Examples section which follows, which is capable ofspecifically binding to the polypeptide set forth by SEQ ID NO:3.

As used herein, the term “epitope” refers to any antigenic determinanton an antigen to which the paratope of an antibody binds.

Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or carbohydrate side chainsand usually have specific three-dimensional structural characteristics,as well as specific charge characteristics.

The term “antibody” as used in this invention includes intact moleculesas well as functional fragments thereof, such as Fab, F(ab′)2, and Fvthat are capable of binding to macrophages. These functional antibodyfragments are defined as follows: (1) Fab, the fragment which contains amonovalent antigen-binding fragment of an antibody molecule, can beproduced by digestion of whole antibody with the enzyme papain to yieldan intact light chain and a portion of one heavy chain; (2) Fab′, thefragment of an antibody molecule that can be obtained by treating wholeantibody with pepsin, followed by reduction, to yield an intact lightchain and a portion of the heavy chain; two Fab′ fragments are obtainedper antibody molecule; (3) (Fab′)₂, the fragment of the antibody thatcan be obtained by treating whole antibody with the enzyme pepsinwithout subsequent reduction; F(ab′)2 is a dimer of two Fab′ fragmentsheld together by two disulfide bonds; (4) Fv, defined as a geneticallyengineered fragment containing the variable region of the light chainand the variable region of the heavy chain expressed as two chains; and(5) Single chain antibody (“SCA”), a genetically engineered moleculecontaining the variable region of the light chain and the variableregion of the heavy chain, linked by a suitable polypeptide linker as agenetically fused single chain molecule.

Methods of producing polyclonal and monoclonal antibodies as well asfragments thereof are well known in the art (See for example, Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,New York, 1988, incorporated herein by reference and the Material andExperimental Methods section of Example 3 in the Examples section whichfollows).

Antibody fragments according to the present invention can be prepared byproteolytic hydrolysis of the antibody or by expression in E. coli ormammalian cells (e.g. Chinese hamster ovary cell culture or otherprotein expression systems) of DNA encoding the fragment. Antibodyfragments can be obtained by pepsin or papain digestion of wholeantibodies by conventional methods. For example, antibody fragments canbe produced by enzymatic cleavage of antibodies with pepsin to provide a5S fragment denoted F(ab′)2. This fragment can be further cleaved usinga thiol reducing agent, and optionally a blocking group for thesulfhydryl groups resulting from cleavage of disulfide linkages, toproduce 3.5S Fab′ monovalent fragments. Alternatively, an enzymaticcleavage using pepsin produces two monovalent Fab′ fragments and an Fcfragment directly. These methods are described, for example, byGoldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and referencescontained therein, which patents are hereby incorporated by reference intheir entirety. See also Porter, R. R. [Biochem. J. 73: 119-126 (1959)].Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

Fv fragments comprise an association of VH and VL chains. Thisassociation may be noncovalent, as described in Inbar et al. [Proc.Nat'l Acad. Sci. USA 69:2659-62 (19720]. Alternatively, the variablechains can be linked by an intermolecular disulfide bond or cross-linkedby chemicals such as glutaraldehyde. Preferably, the Fv fragmentscomprise VH and VL chains connected by a peptide linker. Thesesingle-chain antigen binding proteins (sFv) are prepared by constructinga structural gene comprising DNA sequences encoding the VH and VLdomains connected by an oligonucleotide. The structural gene is insertedinto an expression vector, which is subsequently introduced into a hostcell such as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing sFvs are described, for example, by [Whitlow andFilpula, Methods 2: 97-105 (1991); Bird et al., Science 242:423-426(1988); Pack et al., Bio/Technology 11:1271-77 (1993); and U.S. Pat. No.4,946,778, which is hereby incorporated by reference in its entirety.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells. See, for example, Larrick and Fry[Methods, 2: 106-10 (1991)].

Humanized forms of non-human (e.g., murine) antibodies are chimericmolecules of immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab′).sub.2 or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromnon-human immunoglobulin. Humanized antibodies include humanimmunoglobulins (recipient antibody) in which residues form acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin Hones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as import residues, which aretypically taken from an import variable domain. Humanization can beessentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such humanized antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly,human antibodies can be made by introduction of human immunoglobulinloci into transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10: 779-783(1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14: 826 (1996); and Lonberg and Huszar,Intern. Rev. Immunol. 13, 65-93 (1995). It will be appreciated thattargeting of particular compartment within the cell can be achievedusing intracellular antibodies (also known as “intrabodies”). These areessentially SCA to which intracellular localization signals have beenadded (e.g., ER, mitochondrial, nuclear, cytoplasmic). This technologyhas been successfully applied in the art (for review, see Richardson andMarasco, 1995, TIBTECH vol. 13). Intrabodies have been shown tovirtually eliminate the expression of otherwise abundant cell surfacereceptors and to inhibit a protein function within a cell (See, forexample, Richardson et al., 1995, Proc. Natl. Acad. Sci. USA 92:3137-3141; Deshane et al., 1994, Gene Ther. 1: 332-337; Marasco et al.,1998 Human Gene Ther 9: 1627-42; Shaheen et al., 1996 J. Virol. 70:3392-400; Werge, T. M. et al., 1990, FEBS Letters 274:193-198; Carlson,J. R. 1993 Proc. Natl. Acad. Sci. USA 90:7427-7428; Biocca, S. et al.,1994, Bio/Technology 12: 396-399; Chen, S-Y. et al., 1994, Human GeneTherapy 5:595-601; Duan, L et al., 1994, Proc. Natl. Acad. Sci. USA91:5075-5079; Chen, S-Y. et al., 1994, Proc. Natl. Acad. Sci. USA91:5932-5936; Beerli, R. R. et al., 1994, J. Biol. Chem.269:23931-23936; Mhashilkar, A. M. et al., 1995, EMBO J. 14:1542-1551;PCT Publication No. WO 94/02610 by Marasco et al.; and PCT PublicationNo. WO 95/03832 by Duan et al.).

To prepare an intracellular antibody expression vector, the cDNAencoding the antibody light and heavy chains specific for the targetprotein of interest are isolated, typically from a hybridoma thatsecretes a monoclonal antibody specific for the marker. Hybridomassecreting anti-marker monoclonal antibodies, or recombinant monoclonalantibodies, can be prepared using methods known in the art. Once amonoclonal antibody specific for the marker protein is identified (e.g.,either a hybridoma-derived monoclonal antibody or a recombinant antibodyfrom a combinatorial library), DNAs encoding the light and heavy chainsof the monoclonal antibody are isolated by standard molecular biologytechniques. For hybridoma derived antibodies, light and heavy chaincDNAs can be obtained, for example, by PCR amplification or cDNA libraryscreening. For recombinant antibodies, such as from a phage displaylibrary, cDNA encoding the light and heavy chains can be recovered fromthe display package (e.g., phage) isolated during the library screeningprocess and the nucleotide sequences of antibody light and heavy chaingenes are determined. For example, many such sequences are disclosed inKabat, E. A., et al. (1991) Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242 and in the “Vbase” human germline sequencedatabase. Once obtained, the antibody light and heavy chain sequencesare cloned into a recombinant expression vector using standard methods.

As is shown in FIGS. 23-27 and is described in Example 7 of the Examplessection which follows, both the MSF-A and HIF-1α are co-localized in thecell nucleus. To direct the specific expression of an antibody to thecell nuclei, a nuclear localization signal coding sequence (e.g.,PKKKRKV; Eguchi A, et al., 2005, J. Control Release. 104: 507-19) ispreferably ligated to a nucleic acid construct encoding the antibody,preferably, downstream of the coding sequence of the antibody. Anintracellular antibody expression vector can encode an intracellularantibody in one of several different forms. For example, in oneembodiment, the vector encodes full-length antibody light and heavychains such that a full-length antibody is expressed intracellularly. Inanother embodiment, the vector encodes a full-length light chain butonly the VH/CH1 region of the heavy chain such that a Fab fragment isexpressed intracellularly. In another embodiment, the vector encodes asingle chain antibody (scFv) wherein the variable regions of the lightand heavy chains are linked by a flexible peptide linker [e.g.,(Gly₄Ser)₃ and expressed as a single chain molecule. To inhibit markeractivity in a cell, the expression vector encoding the intracellularantibody is introduced into the cell by standard transfection methods,as discussed hereinbefore.

Another agent capable of downregulating MSF-A-dependent HIF-α activity,or preventing the formation of an MSF-A-HIF-1α complex is a smallinterfering RNA (siRNA) molecule which is capable of downregulatingexpression of MSF-A or HIF-1α. RNA interference is a two step process.The first step, which is termed as the initiation step, input dsRNA isdigested into 21-23 nucleotide (nt) small interfering RNAs (siRNA),probably by the action of Dicer, a member of the RNase III family ofdsRNA-specific ribonucleases, which processes (cleaves) dsRNA(introduced directly or via a transgene or a virus) in an ATP-dependentmanner. Successive cleavage events degrade the RNA to 19-21 by duplexes(siRNA), each with 2-nucleotide 3′ overhangs [Hutvagner and Zamore Curr.Opin. Genetics and Development 12:225-232 (2002); and Bernstein Nature409:363-366 (2001)]. In the effector step, the siRNA duplexes bind to anuclease complex to from the RNA-induced silencing complex (RISC). AnATP-dependent unwinding of the siRNA duplex is required for activationof the RISC. The active RISC then targets the homologous transcript bybase pairing interactions and cleaves the mRNA into 12 nucleotidefragments from the 3′ terminus of the siRNA [Hutvagner and Zamore Curr.Opin. Genetics and Development 12:225-232 (2002); Hammond et al. (2001)Nat. Rev. Gen. 2:110-119 (2001); and Sharp Genes. Dev. 15:485-90(2001)]. Although the mechanism of cleavage is still to be elucidated,research indicates that each RISC contains a single siRNA and an RNase[Hutvagner and Zamore Curr. Opin. Genetics and Development 12:225-232(2002)].

Because of the remarkable potency of RNAi, an amplification step withinthe RNAi pathway has been suggested. Amplification could occur bycopying of the input dsRNAs which would generate more siRNAs, or byreplication of the siRNAs formed. Alternatively or additionally,amplification could be effected by multiple turnover events of the RISC[Hammond et al. Nat. Rev. Gen. 2:110-119 (2001), Sharp Genes. Dev.15:485-90 (2001); Hutvagner and Zamore Cum Opin. Genetics andDevelopment 12:225-232 (2002)]. For more information on RNAi see thefollowing reviews Tuschl ChemBiochem. 2:239-245 (2001); Cullen Nat.Immunol. 3:597-599 (2002); and Brantl Biochem. Biophys. Act. 1575:15-25(2002).

Synthesis of RNAi molecules suitable for use with the present inventioncan be effected as follows. First, the MSF-A and/or HIF-1α mRNA sequenceis scanned downstream of the AUG start codon for AA dinucleotidesequences. Occurrence of each AA and the 3′ adjacent 19 nucleotides isrecorded as potential siRNA target sites. Preferably, siRNA target sitesare selected from the open reading frame, as untranslated regions (UTRs)are richer in regulatory protein binding sites. UTR-binding proteinsand/or translation initiation complexes may interfere with binding ofthe siRNA endonuclease complex [Tuschl, T. 2001, ChemBiochem.2:239-245]. It will be appreciated though, that siRNAs directed atuntranslated regions may also be effective, as demonstrated for GAPDHwherein siRNA directed at the 5′ UTR mediated about 90% decrease incellular GAPDH mRNA and completely abolished protein level(www.ambion.com/techlib/tn/91/912.html).

Second, potential target sites are compared to an appropriate genomicdatabase (e.g., human, mouse, rat etc.) using any sequence alignmentsoftware, such as the BLAST software available from the NCBI server(www.ncbi.nlm.nih.gov/BLAST/). Putative target sites which exhibitsignificant homology to other coding sequences are filtered out.

Qualifying target sequences are selected as template for siRNAsynthesis. Preferred sequences are those including low G/C content asthese have proven to be more effective in mediating gene silencing ascompared to those with G/C content higher than 55%. Several target sitesare preferably selected along the length of the target gene forevaluation. For better evaluation of the selected siRNAs, a negativecontrol is preferably used in conjunction. Negative control siRNApreferably include the same nucleotide composition as the siRNAs butlack significant homology to the genome. Thus, a scrambled nucleotidesequence of the siRNA is preferably used, provided it does not displayany significant homology to any other gene.

Suitable anti-MSF-A siRNAs can be for example the5′-GCCUCCUGAGUAAGACUUCtt (SEQ ID NO:4194) or the5′-CGUGCCUCCUGAGUAAGACtt (SEQ ID NO:4195) siRNA sequences.

Another agent capable of downregulating MSF-A-dependent HIF-α activity,or preventing the formation of an MSF-A-HIF-1α complex is a DNAzymemolecule capable of specifically cleaving an mRNA transcript or DNAsequence of the MSF-A and/or HIF-1α. DNAzymes are single-strandedpolynucleotides which are capable of cleaving both single and doublestranded target sequences (Breaker, R. R. and Joyce, G. Chemistry andBiology 1995; 2:655; Santoro, S. W. & Joyce, G. F. Proc. Natl, Acad.Sci. USA 1997; 943:4262). A general model (the “10-23” model) for theDNAzyme has been proposed. “10-23” DNAzymes have a catalytic domain of15 deoxyribonucleotides, flanked by two substrate-recognition domains ofseven to nine deoxyribonucleotides each. This type of DNAzyme caneffectively cleave its substrate RNA at purine:pyrimidine junctions(Santoro, S. W. & Joyce, G. F. Proc. Natl, Acad. Sci. USA 199; for revof DNAzymes see Khachigian, L M [Curr Opin Mol Ther 4:119-21 (2002)].

Examples of construction and amplification of synthetic, engineeredDNAzymes recognizing single and double-stranded target cleavage siteshave been disclosed in U.S. Pat. No. 6,326,174 to Joyce et al. DNAzymesof similar design directed against the human Urokinase receptor wererecently observed to inhibit Urokinase receptor expression, andsuccessfully inhibit colon cancer cell metastasis in vivo (Itoh et al,20002, Abstract 409, Ann Meeting Am Soc Gen Ther. www.asgt.org). Inanother application, DNAzymes complementary to bcr-abl oncogenes weresuccessful in inhibiting the oncogenes expression in leukemia cells, andlessening relapse rates in autologous bone marrow transplant in cases ofCML and ALL.

Downregulation of MSF-A or HIF-1α can also be effected by using anantisense polynucleotide capable of specifically hybridizing with anmRNA transcript encoding the MSF-A and/or HIF-1α and thus preventing theassociation between MSF-A and HIF-1α.

Design of antisense molecules which can be used to efficientlydown-regulate MSF-A or HIF-1α must be effected while considering twoaspects important to the antisense approach. The first aspect isdelivery of the oligonucleotide into the cytoplasm of the appropriatecells, while the second aspect is design of an oligonucleotide whichspecifically binds the designated mRNA within cells in a way whichinhibits translation thereof.

The prior art teaches of a number of delivery strategies which can beused to efficiently deliver oligonucleotides into a wide variety of celltypes [see, for example, Luft J Mol Med 76: 75-6 (1998); Kronenwett etal. Blood 91: 852-62 (1998); Rajur et al. Bioconjug Chem 8: 935-40(1997); Lavigne et al. Biochem Biophys Res Commun 237: 566-71 (1997) andAoki et al. (1997) Biochem Biophys Res Commun 231: 540-5 (1997)].

In addition, algorithms for identifying those sequences with the highestpredicted binding affinity for their target mRNA based on athermodynamic cycle that accounts for the energetics of structuralalterations in both the target mRNA and the oligonucleotide are alsoavailable [see, for example, Walton et al. Biotechnol Bioeng 65: 1-9(1999)].

Such algorithms have been successfully used to implement an antisenseapproach in cells. For example, the algorithm developed by Walton et al.enabled scientists to successfully design antisense oligonucleotides forrabbit beta-globin (RBG) and mouse tumor necrosis factor-alpha (TNFalpha) transcripts. The same research group has more recently reportedthat the antisense activity of rationally selected oligonucleotidesagainst three model target mRNAs (human lactate dehydrogenase A and Band rat gp130) in cell culture as evaluated by a kinetic PCR techniqueproved effective in almost all cases, including tests against threedifferent targets in two cell types with phosphodiester andphosphorothioate oligonucleotide chemistries.

In addition, several approaches for designing and predicting efficiencyof specific oligonucleotides using an in vitro system were alsopublished (Matveeva et al., Nature Biotechnology 16: 1374-1375 (1998)].

Suitable antisense oligonucleotides which can be utilized todown-regulate MSF-A or HIF-1α expression are exemplified by5′-GCTCCCTCCAACCAGACTCA-3′ (SEQ ID NO:4196) or5′-GGGTTCTTTGCTTCTGTGTC-3′ (SEQ ID NO:4197), respectively.

Several clinical trials have demonstrated safety, feasibility andactivity of antisense oligonucleotides. For example, antisenseoligonucleotides suitable for the treatment of cancer have beensuccessfully used [Holmund et al., Curr Opin Mol Ther 1:372-85 (1999)],while treatment of hematological malignancies via antisenseoligonucleotides targeting c-myb gene, p53 and Bcl-2 had enteredclinical trials and had been shown to be tolerated by patients [GerwitzCurr Opin Mol Ther 1:297-306 (1999)].

More recently, antisense-mediated suppression of human heparanase geneexpression has been reported to inhibit pleural dissemination of humancancer cells in a mouse model [Uno et al., Cancer Res 61:7855-60(2001)].

Thus, the current consensus is that recent developments in the field ofantisense technology which, as described above, have led to thegeneration of highly accurate antisense design algorithms and a widevariety of oligonucleotide delivery systems, enable an ordinarilyskilled artisan to design and implement antisense approaches suitablefor downregulating expression of known sequences without having toresort to undue trial and error experimentation.

Another agent capable of downregulating MSF-A-dependent HIF-α activity,or preventing the formation of an MSF-A-HIF-1α complex is a ribozymemolecule capable of specifically cleaving an mRNA transcript encodingMSF-A or HIF-1α. Ribozymes are being increasingly used for thesequence-specific inhibition of gene expression by the cleavage of mRNAsencoding proteins of interest [Welch et al., Curr Opin Biotechnol.9:486-96 (1998)]. The possibility of designing ribozymes to cleave anyspecific target RNA has rendered them valuable tools in both basicresearch and therapeutic applications. In the therapeutics area,ribozymes have been exploited to target viral RNAs in infectiousdiseases, dominant oncogenes in cancers and specific somatic mutationsin genetic disorders [Welch et al., Clin Diagn Virol. 10:163-71 (1998)].Most notably, several ribozyme gene therapy protocols for HIV patientsare already in Phase 1 trials. More recently, ribozymes have been usedfor transgenic animal research, gene target validation and pathwayelucidation. Several ribozymes are in various stages of clinical trials.ANGIOZYME was the first chemically synthesized ribozyme to be studied inhuman clinical trials. ANGIOZYME specifically inhibits formation of theVEGF-r (Vascular Endothelial Growth Factor receptor), a key component inthe angiogenesis pathway. Ribozyme Pharmaceuticals, Inc., as well asother firms have demonstrated the importance of anti-angiogenesistherapeutics in animal models. HEPTAZYME, a ribozyme designed toselectively destroy Hepatitis C Virus (HCV) RNA, was found effective indecreasing Hepatitis C viral RNA in cell culture assays (RibozymePharmaceuticals, Incorporated—WEB home page).

Another agent capable of downregulating MSF-A-dependent HIF-1α activity,preventing the formation of an MSF-A-HIF-1α complex or destabilizing analready formed complex can be any molecule which binds to and/or cleavesMSF-A or HIF-1α (e.g., antagonists, or inhibitory peptides) or preventsMSF-A or HIF-1α activation or substrate binding. An example of such amolecule is a non-functional MSF-A polypeptide and/or a non-functionalHIF-1α polypeptide.

As used herein, the phrases “non-functional MSF-A polypeptide” and/or a“non-functional HIF-1α polypeptide” refer to any polypeptide lacking atleast one function of the MSF-A and/or HIF-1α polypeptides, including,but not limited to, substrate binding or interaction with otherproteins. Such a polypeptide can include at least one insertion,deletion or substitution of an amino acid which results in an alteredfunction of the MSF-A and/or the HIF-1α proteins. Non-limiting examplesof non-functional MSF-A polypeptides are the N-terminal deleted form ofthe MSF-A protein as set forth by SEQ ID NO:10 which is encoded by SEQID NO:7 and/or the GTP-binding site deleted form of the MSF-A protein asset forth by SEQ ID NO:4215.

It will be appreciated that a non-functional analogue of at least acatalytic or binding portion of either MSF-A or HIF-1α can be also usedto prevent the formation of the MSF-A-HIF-1α protein complex. Forexample, an MSF-A analogue consisting of at least one substituted,inserted or deleted amino acid at the N-terminal of MSF-A (i.e., any ofthe first 25 amino acids as set forth in SEQ ID NO:3) can be used toprevent MSF-A-dependent activation of HIF-1α.

Such non-functional MSF-A or HIF-1α polypeptides can be utilized per seor can be expressed in cells by ligating a polynucleotide encoding thenon-functional MSF-A or HIF-1α polypeptide into an expression vector asis further described hereinbelow.

In addition to the non-functional polypeptides described above, thepresent invention can also employ peptides, peptide analogues ormimetics thereof which are derived from either the HIF-1α or the MSF-Aand which are capable of preventing the formation of, or dissociating,the MSF-A-HIF-1α protein complex.

Such peptides, peptide analogues or mimetics thereof are preferablyshort amino acid sequences of at least 2 or 3 amino acids, preferably atleast 4, more preferably, at least 5, more preferably, in the range of5-30, even more preferably in the range of 5-25 amino acids which arederived from either the HIF-1α or the MSF-A proteins. A non-limitingexample of such a peptide can be the 25 mer peptide derived from theunique N-terminal sequence of MSF-A (SEQ ID NO:3). The amino acidsequence of such a peptide is: Met Lys Lys Ser Tyr Ser Gly Gly Thr ArgThr Ser Ser Gly Arg Leu Arg Arg Leu Gly Asp Ser Ser Gly Pro as set forthby SEQ ID NO:4213.

As used herein the term “mimetics” refers to molecular structures, whichserve as substitutes for the peptide of the present invention inprevention of the formation of or dissociation of the MSF-A-HIF-1αprotein complex (Morgan et al. (1989) Ann. Reports Med. Chem. 24:243-252for a review of peptide mimetics). Peptide mimetics, as used herein,include synthetic structures (known and yet unknown), which may or maynot contain amino acids and/or peptide bonds, but retain the structuraland functional features of preventing the formation of or dissociatingthe HIF-1α-MSF-A protein complex. Types of amino acids which can beutilized to generate mimetics are further described hereinbelow. Theterm, “peptide mimetics” also includes peptoids and oligopeptoids, whichare peptides or oligomers of N-substituted amino acids [Simon et al.(1972) Proc. Natl. Acad. Sci. USA 89:9367-9371]. Further included aspeptide mimetics are peptide libraries, which are collections ofpeptides designed to be of a given amino acid length and representingall conceivable sequences of amino acids corresponding thereto.Non-limiting examples of such peptide libraries are provided in SEQ IDNOs:12-2462 or 2463-4193 for peptides derived from HIF-1α or MSF-A,respectively. Methods of producing peptide mimetics are describedhereinbelow.

The term “peptide” as used herein encompasses native peptides (eitherdegradation products, synthetically synthesized peptides or recombinantpeptides) and as mentioned hereinabove, peptidomimetics (typically,synthetically synthesized peptides), as well as peptoids andsemipeptoids which are peptide analogs, which may have, for example,modifications rendering the peptides more stable while in a body or morecapable of penetrating into cells. Such modifications include, but arenot limited to N terminus modification, C terminus modification, peptidebond modification, including, but not limited to, CH2-NH, CH2-S,CH2-S═O, O═C—NH, CH2-O, CH2-CH2, S═C—NH, CH═CH or CF═CH, backbonemodifications, and residue modification. Methods for preparingpeptidomimetic compounds are well known in the art and are specified,for example, in Quantitative Drug Design, C. A. Ramsden Gd., Chapter17.2, F. Choplin Pergamon Press (1992), which is incorporated byreference as if fully set forth herein. Further details in this respectare provided hereinunder.

Peptide bonds (—CO—NH—) within the peptide may be substituted, forexample, by N-methylated bonds (—N(CH3)—CO—), ester bonds(—C(R)H—C—O—O—C(R)—N—), ketomethylen bonds (—CO—CH2—), a-aza bonds(—NH—N(R)—CO—), wherein R is any alkyl, e.g., methyl, carba bonds(—CH2—NH—), hydroxyethylene bonds (—CH(OH)—CH2—), thioamide bonds(—CS—NH—), olefinic double bonds (—CH═CH—), retro amide bonds (—NH—CO—),peptide derivatives (—N(R)—CH2—CO—), wherein R is the “normal” sidechain, naturally presented on the carbon atom.

These modifications can occur at any of the bonds along the peptidechain and even at several (2-3) at the same time.

Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted forsynthetic non-natural acid such as Phenylglycine, TIC, naphthylelanine(Nol), ring-methylated derivatives of Phe, halogenated derivatives ofPhe or o-methyl-Tyr.

In addition to the above, the peptides of the present invention may alsoinclude one or more modified amino acids or one or more non-amino acidmonomers (e.g. fatty acids, complex carbohydrates etc).

As used herein in the specification the term “amino acid” or “aminoacids” is understood to include the 20 naturally occurring amino acids;those amino acids often modified post-translationally in vivo,including, for example, hydroxyproline, phosphoserine andphosphothreonine; and other unusual amino acids including, but notlimited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine,nor-leucine and ornithine. Furthermore, the term “amino acid” includesboth D- and L-amino acids.

Tables 1 and 2 below list naturally occurring amino acids (Table 1) andnon-conventional or modified amino acids (Table 2) which can be usedwith the present invention.

TABLE 1 Amino Acid Three-Letter Abbreviation One-letter Symbol alanineAla A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys CGlutamine Gln Q Glutamic Acid Glu E glycine Gly G Histidine His Hisoleucine Iie I leucine Leu L Lysine Lys K Methionine Met Mphenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr Ttryptophan Trp W tyrosine Tyr Y Valine Val V Any amino acid as Xaa Xabove

TABLE 2 Non-conventional amino acid Code Non-conventional amino acidCode α-aminobutyric acid Abu L-N-methylalanine Nmalaα-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmargaminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylateL-N-methylaspartic acid Nmasp aminoisobutyric acid AibL-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgincarboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine ChexaL-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucineNmile D-alanine Dal L-N-methylleucine Nmleu D-arginine DargL-N-methyllysine Nmlys D-aspartic acid Dasp L-N-methylmethionine NmmetD-cysteine Dcys L-N-methylnorleucine Nmnle D-glutamine DglnL-N-methylnorvaline Nmnva D-glutamic acid Dglu L-N-methylornithine NmornD-histidine Dhis L-N-methylphenylalanine Nmphe D-isoleucine DileL-N-methylproline Nmpro D-leucine Dleu L-N-methylserine Nmser D-lysineDlys L-N-methylthreonine Nmthr D-methionine Dmet L-N-methyltryptophanNmtrp D-ornithine Dorn L-N-methyltyrosine Nmtyr D-phenylalanine DpheL-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycine NmetgD-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine DthrL-norleucine Nle D-tryptophan Dtrp L-norvaline Nva D-tyrosine Dtyrα-methyl-aminoisobutyrate Maib D-valine Dval α-methyl-γ-aminobutyrateMgabu D-α-methylalanine Dmala α-methylcyclohexylalanine MchexaD-α-methylarginine Dmarg α-methylcyclopentylalanine McpenD-α-methylasparagine Dmasn α-methyl-α-napthylalanine ManapD-α-methylaspartate Dmasp α-methylpenicillamine Mpen D-α-methylcysteineDmcys N-(4-aminobutyl)glycine Nglu D-α-methylglutamine DmglnN-(2-aminoethyl)glycine Naeg D-α-methylhistidine DmhisN-(3-aminopropyl)glycine Norn D-α-methylisoleucine DmileN-amino-α-methylbutyrate Nmaabu D-α-methylleucine Dmleu α-napthylalanineAnap D-α-methyllysine Dmlys N-benzylglycine Nphe D-α-methylmethionineDmmet N-(2-carbamylethyl)glycine Ngln D-α-methylornithine DmornN-(carbamylmethyl)glycine Nasn D-α-methylphenylalanine DmpheN-(2-carboxyethyl)glycine Nglu D-α-methylproline DmproN-(carboxymethyl)glycine Nasp D-α-methylserine Dmser N-cyclobutylglycineNcbut D-α-methylthreonine Dmthr N-cycloheptylglycine NchepD-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex D-α-methyltyrosineDmty N-cyclodecylglycine Ncdec D-α-methylvaline DmvalN-cyclododeclglycine Ncdod D-α-methylalnine Dnmala N-cyclooctylglycineNcoct D-α-methylarginine Dnmarg N-cyclopropylglycine NcproD-α-methylasparagine Dnmasn N-cycloundecylglycine NcundD-α-methylasparatate Dnmasp N-(2,2-diphenylethyl)glycine NbhmD-α-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine NbheD-N-methylleucine Dnmleu N-(3-indolylyethyl) glycine NhtrpD-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate NmgabuN-methylcyclohexylalanine Nmchexa D-N-methylmethionine DnmmetD-N-methylornithine Dnmorn N-methylcyclopentylalanine NmcpenN-methylglycine Nala D-N-methylphenylalanine DnmpheN-methylaminoisobutyrate Nmaib D-N-methylproline DnmproN-(1-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nleu D-N-methylthreonine DnmthrD-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine NvaD-N-methyltyrosine Dnmtyr N-methyla-napthylalanine NmanapD-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acidGabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine TbugN-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine PenL-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine MargL-α-methylasparagine Masn L-α-methylaspartate MaspL-α-methyl-t-butylglycine Mtbug L-α-methylcysteine McysL-methylethylglycine Metg L-α-methylglutamine Mgln L-α-methylglutamateMglu L-α-methylhistidine Mhis L-α-methylhomo phenylalanine MhpheL-α-methylisoleucine Mile N-(2-methylthioethyl)glycine NmetD-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine NargD-N-methylglutamate Dnmglu N-(1-hydroxyethyl)glycine NthrD-N-methylhistidine Dnmhis N-(hydroxyethyl)glycine NserD-N-methylisoleucine Dnmile N-(imidazolylethyl)glycine NhisD-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine NhtrpD-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate NmgabuN-methylcyclohexylalanine Nmchexa D-N-methylmethionine DnmmetD-N-methylornithine Dnmorn N-methylcyclopentylalanine NmcpenN-methylglycine Nala D-N-methylphenylalanine DnmpheN-methylaminoisobutyrate Nmaib D-N-methylproline DnmproN-(1-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nleu D-N-methylthreonine DnmthrD-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine NvalD-N-methyltyrosine Dnmtyr N-methyla-napthylalanine NmanapD-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acidGabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine TbugN-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine PenL-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine MargL-α-methylasparagine Masn L-α-methylaspartate MaspL-α-methyl-t-butylglycine Mtbug L-α-methylcysteine McysL-methylethylglycine Metg L-α-methylglutamine Mgln L-α-methylglutamateMglu L-α-methylhistidine Mhis L-α-methylhomophenylalanine MhpheL-α-methylisoleucine Mile N-(2-methylthioethyl)glycine NmetL-α-methylleucine Mleu L-α-methyllysine Mlys L-α-methylmethionine MmetL-α-methylnorleucine Mnle L-α-methylnorvaline Mnva L-α-methylornithineMorn L-α-methylphenylalanine Mphe L-α-methylproline MproL-α-methylserine mser L-α-methylthreonine Mthr L-α-methylvaline MtrpL-α-methyltyrosine Mtyr L-α-methylleucine Mval NnbhmL-N-methylhomophenylalanine Nmhphe N-(N-(2,2-diphenylethyl)N-(N-(3,3-diphenylpropyl) carbamylmethyl-glycine Nnbhmcarbamylmethyl(1)glycine Nnbhe 1-carboxy-1-(2,2-diphenyl Nmbcethylamino)cyclopropane

Since the present peptides are preferably utilized in therapeutics ordiagnostics which require the peptides to be in soluble form, thepeptides of the present invention preferably include one or morenon-natural or natural polar amino acids, including but not limited toserine and threonine which are capable of increasing peptide solubilitydue to their hydroxyl-containing side chain.

The peptides of the present invention are preferably utilized in alinear form, although it will be appreciated that in cases wherecyclicization does not severely interfere with peptide characteristics,cyclic forms of the peptide can also be utilized.

The peptides of present invention can be biochemically synthesized suchas by using standard solid phase techniques. These methods includeexclusive solid phase synthesis, partial solid phase synthesis methods,fragment condensation and classical solution synthesis. These methodsare preferably used when the peptide is relatively short (i.e., 10 kDa)and/or when it cannot be produced by recombinant techniques (i.e., notencoded by a nucleic acid sequence) and therefore involve differentchemistry.

Solid phase peptide synthesis procedures are well known in the art andfurther described by John Morrow Stewart and Janis Dillaha Young, SolidPhase Peptide Syntheses (2nd Ed., Pierce Chemical Company, 1984).

Synthetic peptides can be purified by preparative high performanceliquid chromatography [Creighton T. (1983) Proteins, structures andmolecular principles. WH Freeman and Co. N.Y.] and the composition ofwhich can be confirmed via amino acid sequencing.

In cases where large amounts of the peptides of the present inventionare desired, the peptides of the present invention can be generatedusing recombinant techniques such as described by Bitter et al., (1987)Methods in Enzymol. 153:516-544, Studier et al. (1990) Methods inEnzymol. 185:60-89, Brisson et al. (1984) Nature 310:511-514, Takamatsuet al. (1987) EMBO J. 6:307-311, Coruzzi et al. (1984) EMBO J.3:1671-1680, Brogli et al., (1984) Science 224:838-843, Gurley et al.(1986) Mol. Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988,Methods for Plant Molecular Biology, Academic Press, NY, Section VIII,pp 421-463.

Generation of peptide mimetics, as described hereinabove, is effectedusing various approaches, including, for example, display techniques,using a plurality of display vehicles (such as phages, viruses orbacteria) each displaying a short peptide sequence as describedhereinabove. For example, a display library containing peptides derivedfrom HIF-1α or MSF-A (as set forth by SEQ ID NOs:12-2462 or 2463-4193,respectively) can be screened with MSF-A or HIF-1α (respectively) inorder to identify peptides capable of binding one or both constituentsof this protein complex. Such peptides would be potentially capable ofpreventing the formation of the complex or capable of destabilizing thecomplex.

Methods of constructing and screening peptide display libraries are wellknown in the art. Such methods are described in, for example, Young A C,et al., “The three-dimensional structures of a polysaccharide bindingantibody to Cryptococcus neoformans and its complex with a peptide froma phage display library: implications for the identification of peptidemimotopes” J Mol Biol 1997 Dec. 12; 274(4):622-34; Giebel L B et al.“Screening of cyclic peptide phage libraries identifies ligands thatbind streptavidin with high affinities” Biochemistry 1995 Nov. 28;34(47):15430-5; Davies E L et al., “Selection of specific phage-displayantibodies using libraries derived from chicken immunoglobulin genes” JImmunol Methods 1995 Oct. 12; 186(1):125-35; Jones C RT al. “Currenttrends in molecular recognition and bioseparation” J Chromatogr A 1995Jul. 14; 707(1):3-22; Deng S J et al. “Basis for selection of improvedcarbohydrate-binding single-chain antibodies from synthetic genelibraries” Proc Natl Acad Sci USA 1995 May 23; 92(11):4992-6; and Deng SJ et al. “Selection of antibody single-chain variable fragments withimproved carbohydrate binding by phage display” J Biol Chem 1994 Apr. 1;269(13):9533-8, which are incorporated herein by reference.

Peptide mimetics can also be uncovered using computational biology. Forexample, various compounds can be computationally analyzed for anability to prevent the formation of or dissociate the MSF-A-HIF-1αprotein complex using a variety of three-dimensional computationaltools. Software programs useful for displaying three-dimensionalstructural models, such as RIBBONS (Carson, M., 1997. Methods inEnzymology 277, 25), 0 (Jones, T A. et al., 1991. Acta Crystallogr. A47,110), DINO (DINO: Visualizing Structural Biology (2001)http://www.dino3d.org); and QUANTA, INSIGHT, SYBYL, MACROMODE, ICM,MOLMOL, RASMOL and GRASP (reviewed in Kraulis, J., 1991. ApplCrystallogr. 24, 946) can be utilized to model interactions between theMSF-A-HIF-1α protein complex and prospective peptide mimetics to therebyidentify peptides which display the highest probability of binding andinterfering of the association between MSF-A and HIF-1α. Computationalmodeling of protein-peptide interactions has been successfully used inrational drug design, for further detail, see Lam et al., 1994. Science263, 380; Wlodawer et al., 1993. Ann Rev Biochem. 62, 543; Appelt, 1993.Perspectives in Drug Discovery and Design 1, 23; Erickson, 1993.Perspectives in Drug Discovery and Design 1, 109, and Mauro M J. et al.,2002. J Clin Oncol. 20, 325-34.

Aptamers are nucleic acids or oligonucleotide molecules, typically of10-15 kDa in size (30-45 nucleotides) which are capable of specificallybinding to selected targets and altering their activity.

Aptamers may be double-stranded or single-stranded, and may includedeoxyribonucleotides, ribonucleotides, nucleotide derivatives, or othernucleotide-like molecules. The nucleotide components of an aptamer mayhave modified sugar groups (e.g., the 2′-OH group of a ribonucleotidemay be replaced by 2′-F or 2′—NH2), which may improve a desiredproperty, e.g., resistance to nucleases or longer lifetime in vivo.Aptamers may be conjugated to other molecules, e.g., a high molecularweight carrier to slow the clearance of the aptamer from the circulatorysystem, or they can be specifically cross-linked to their cognateligands, e.g., by photo-activation of a cross-linker (See, e.g., Brody,E. N. and L. Gold (2000) J. Biotechnol. 74:5-13.)

Aptamers are produced using in vitro selection processes which allow thespecificity and affinity of the aptamer to be tightly controlled.

A suitable method for generating an aptamer to a target of interest(e.g., the MSF-A, the HIF-1α and/or the MSF-A-HIF1α complex) is the“Systematic Evolution of Ligands by EXponential Enrichment” (SELEX™).The SELEX™ method is described in U.S. patent application Ser. No.07/536,428, filed Jun. 11, 1990, now abandoned, U.S. Pat. No. 5,475,096entitled “Nucleic Acid Ligands”, and U.S. Pat. No. 5,270,163 (see alsoWO 91/19813) entitled “Nucleic Acid Ligands”. Briefly, a mixture ofnucleic acids is contacted with the target molecule under conditionsfavorable for binding. The unbound nucleic acids are partitioned fromthe bound nucleic acids, and the nucleic acid-target complexes aredissociated. Then the dissociated nucleic acids are amplified to yield aligand-enriched mixture of nucleic acids, which is subjected to repeatedcycles of binding, partitioning, dissociating and amplifying as desiredto yield highly specific high affinity nucleic acid ligands to thetarget molecule.

Aptamers have been generated for over 100 proteins including growthfactors, transcription factors, enzymes, immunoglobulins, and receptors.Typical aptamers bind to their targets with sub-nanomolar affinity anddiscriminate against closely related targets (i.e., other proteins fromthe same gene family). A series of structural studies have shown thataptamers are capable of using the same types of binding interactions(hydrogen bonding, electrostatic complementarity, hydrophobic contacts,and steric exclusion) that drive affinity and specificity inantibody-antigen complexes.

Thus, the teachings of the present invention can be used to treat canceror cancerous tumors. Briefly, an agent capable of preventing theformation of or dissociating the MSF-A-HIF-1α protein complex such asthe siRNA set forth by SEQ ID NO:4195 which is designed fordownregulating the MSF-A mRNA is administered to the individual as partof a pharmaceutical composition (as described hereinbelow) along with apharmaceutical acceptable carrier (e.g., calcium carbonate). It shouldbe noted that since siRNA molecules typically have a limited half-lifethe treatment described above is preferably repeated periodically inorder to prevent tumor growth or progression of cancer.

Although as described hereinabove, activation of HIF-1α through theHIF-1α-MSF-A protein complex is associated with cancerous tumors andcancer metastases, there are several clinical conditions in whichactivation of HIF-1α is desired. For example, in the case of acuteischemia, where the oxygen tension decreases (i.e., hypoxia conditions),HIF-1α is stabilized and thus activates transcription of various targetgenes which contribute to angiogenesis. However, such activation istemporary and often can not overcome and correct the damage caused bythe acute ischemia.

While reducing the present invention to practice, the present inventorhas uncovered that upregulation of MSF-A-dependent HIF-1α activityand/or stabilization of the MSF-A-HIF-1α protein complex can be used toactivate HIF-1α and to treat acute ischemia.

Thus, according to another aspect of the present invention there isprovided a method of treating acute ischemia in cells of an individual.

The method is effected by providing to the individual an agent capableof upregulating an MSF-A-dependent HIF-1α activity and/or stabilizing anMSF-A-HIF-1α protein complex in cells of the individual to thereby treatthe acute ischemia.

The term “individual” as used herein encompasses both males and femalesat any age which are at risk to develop ischemic diseases. For example,smokers or individuals with high blood pressure, diabetes,hypercholesterolemia, a coronary disease, cerebral vascular diseases andatherosclerosis.

According to preferred embodiments of the present invention the acuteischemia is a result of stroke or acute myocardial infraction.

As used herein the term “upregulating” refers to increasing theexpression level and/or activity of MSF-A and/or HIF-1α proteins.

The term “stabilizing” refers to increasing the stability of theMSF-A-HIF-1α protein complex, i.e., enabling the protein complex toretain the interactions between the MSF-A and HIF-1α which consist ofthe protein complex. It will be appreciated that stabilization of theMSF-A-HIF-1α protein complex can be achieved, for example, byupregulating the expression level and/or activity of the MSF-A and/orthe HIF-1α proteins.

Upregulation of the expression level and/or activity of MSF-A or HIF-1αcan be effected at the genomic level (i.e., activation of transcriptionvia promoters, enhancers, regulatory elements), at the transcript level(i.e., correct splicing, polyadenylation, activation of translation) orat the protein level (i.e., post-translational modifications,interaction with substrates and the like). Preferably, agents capable ofincreasing the association between MSF-A and HIF-1α can be used alongwith the present invention.

Following is a list of agents capable of upregulating the expressionlevel and/or activity of MSF-A or HIF-1α.

An agent capable of upregulating expression level of MSF-A or HIF-1α maybe an exogenous polynucleotide sequence designed and constructed toexpress at least a functional portion of the MSF-A or HIF-1α proteins.Accordingly, the exogenous polynucleotide sequence may be a DNA or RNAsequence encoding the MSF-A or HIF-1α molecules, capable of forming theMSF-A-HIF-1α protein complex.

The phrase “functional portion” as used herein refers to part of theMSF-A or HIF-1α proteins (i.e., a polypeptide) which exhibits functionalproperties of the enzyme such as binding a substrate or another protein,forming a protein complex and the like. According to preferredembodiments of the present invention the functional portion of MSF-A orHIF-1α is a polypeptide sequence including amino acids 1-586 as setforth in SEQ ID NO:3 or a polypeptide sequence including amino acids1-826 as set forth in SEQ ID NO:11, respectively. Preferably, thefunctional portion of HIF-1α is a polypeptide sequence including aminoacids 37-373, more preferably, amino acids 36-821 as set forth in SEQ IDNO:11.

MSF-A and HIF-1α have been cloned from human, mouse (HIF-1α) and rat(HIF-1α) sources. Thus, coding sequences information for MSF-A and/orHIF-1α is available from several databases including the GenBankdatabase available through http://www.ncbi.nlm.nih.gov/.

To express exogenous MSF-A or HIF-1α in mammalian cells, apolynucleotide sequence encoding MSF-A or HIF-1α (SEQ ID NO:1 or 2,respectively) is preferably ligated into a nucleic acid constructsuitable for mammalian cell expression. Such a nucleic acid constructincludes a promoter sequence for directing transcription of thepolynucleotide sequence in the cell in a constitutive or induciblemanner.

It will be appreciated that the nucleic acid construct of the presentinvention can also utilize MSF-A or HIF-1α homologues which exhibit thedesired activity. Such homologues can be, for example, at least 80%, atleast 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or 100% identical toSEQ ID NO:1 or 2, respectively, as determined using the BestFit softwareof the Wisconsin sequence analysis package, utilizing the Smith andWaterman algorithm, where gap weight equals 50, length weight equals 3,average match equals 10 and average mismatch equals −9.

Constitutive promoters suitable for use with the present invention arepromoter sequences which are active under most environmental conditionsand most types of cells such as the cytomegalovirus (CMV) and Roussarcoma virus (RSV). Inducible promoters suitable for use with thepresent invention include for example the tetracycline-induciblepromoter [Zabala M, et al., Cancer Res. 2004, 64(8): 2799-804].

The nucleic acid construct (also referred to herein as an “expressionvector”) of the present invention includes additional sequences whichrender this vector suitable for replication and integration inprokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors). Inaddition, a typical cloning vectors may also contain a transcription andtranslation initiation sequence, transcription and translationterminator and a polyadenylation signal.

Eukaryotic promoters typically contain two types of recognitionsequences, the TATA box and upstream promoter elements. The TATA box,located 25-30 base pairs upstream of the transcription initiation site,is thought to be involved in directing RNA polymerase to begin RNAsynthesis. The other upstream promoter elements determine the rate atwhich transcription is initiated.

Enhancer elements can stimulate transcription up to 1,000 fold fromlinked homologous or heterologous promoters. Enhancers are active whenplaced downstream or upstream from the transcription initiation site.Many enhancer elements derived from viruses have a broad host range andare active in a variety of tissues. For example, the SV40 early geneenhancer is suitable for many cell types. Other enhancer/promotercombinations that are suitable for the present invention include thosederived from polyoma virus, human or murine cytomegalovirus (CMV), thelong term repeat from various retroviruses such as murine leukemiavirus, murine or Rous sarcoma virus and HIV. See, Enhancers andEukaryotic Expression, Cold Spring Harbor Press, Cold Spring Harbor,N.Y. 1983, which is incorporated herein by reference.

In the construction of the expression vector, the promoter is preferablypositioned approximately the same distance from the heterologoustranscription start site as it is from the transcription start site inits natural setting. As is known in the art, however, some variation inthis distance can be accommodated without loss of promoter function.

Polyadenylation sequences can also be added to the expression vector inorder to increase the efficiency of MSF-A/or HIF-1α mRNA translation.Two distinct sequence elements are required for accurate and efficientpolyadenylation: GU or U rich sequences located downstream from thepolyadenylation site and a highly conserved sequence of six nucleotides,AAUAAA, located 11-30 nucleotides upstream. Termination andpolyadenylation signals that are suitable for the present inventioninclude those derived from SV40.

In addition to the elements already described, the expression vector ofthe present invention may typically contain other specialized elementsintended to increase the level of expression of cloned nucleic acids orto facilitate the identification of cells that carry the recombinantDNA. For example, a number of animal viruses contain DNA sequences thatpromote the extra chromosomal replication of the viral genome inpermissive cell types. Plasmids bearing these viral replicons arereplicated episomally as long as the appropriate factors are provided bygenes either carried on the plasmid or with the genome of the host cell.

The vector may or may not include a eukaryotic replicon. If a eukaryoticreplicon is present, then the vector is amplifiable in eukaryotic cellsusing the appropriate selectable marker. If the vector does not comprisea eukaryotic replicon, no episomal amplification is possible. Instead,the recombinant DNA integrates into the genome of the engineered cell,where the promoter directs expression of the desired nucleic acid.

The expression vector of the present invention can further includeadditional polynucleotide sequences that allow, for example, thetranslation of several proteins from a single mRNA such as an internalribosome entry site (IRES) and sequences for genomic integration of thepromoter-chimeric polypeptide. It will be appreciated that such anexpression vector can include the coding sequence of both MSF-A andHIF-1a to enable translation of both proteins.

Examples for mammalian expression vectors include, but are not limitedto, pcDNA3, pcDNA3.1(+/−), pGL3, pZeoSV2(+/−), pSecTag2, pDisplay,pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1,pNMT41, pNMT81, which are available from Invitrogen, pCI which isavailable from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which areavailable from Strategene, pTRES which is available from Clontech, andtheir derivatives.

Expression vectors containing regulatory elements from eukaryoticviruses such as retroviruses can be also used. SV40 vectors includepSVT7 and pMT2. Vectors derived from bovine papilloma virus includepBV-1MTHA, and vectors derived from Epstein Bar virus include pHEBO, andp2O5. Other exemplary vectors include pMSG, pAV009/A⁺, pMTO10/A⁺,pMAMneo-5, baculovirus pDSVE, and any other vector allowing expressionof proteins under the direction of the SV-40 early promoter, SV-40 laterpromoter, metallothionein promoter, murine mammary tumor virus promoter,Rous sarcoma virus promoter, polyhedrin promoter, or other promotersshown effective for expression in eukaryotic cells.

As described above, viruses are very specialized infectious agents thathave evolved, in many cases, to elude host defense mechanisms.Typically, viruses infect and propagate in specific cell types. Thetargeting specificity of viral vectors utilizes its natural specificityto specifically target predetermined cell types and thereby introduce arecombinant gene into the infected cell. Thus, the type of vector usedby the present invention will depend on the cell type transformed. Theability to select suitable vectors according to the cell typetransformed is well within the capabilities of the ordinary skilledartisan and as such no general description of selection consideration isprovided herein. For example, bone marrow cells can be targeted usingthe human T cell leukemia virus type I (HTLV-I) and kidney cells may betargeted using the heterologous promoter present in the baculovirusAutographa californica nucleopolyhedrovirus (AcMNPV) as described inLiang C Y et al., 2004 (Arch Virol. 149: 51-60).

Recombinant viral vectors are useful for in vivo expression of MSF-A orHIF-1α since they offer advantages such as lateral infection andtargeting specificity. Lateral infection is inherent in the life cycleof, for example, retrovirus and is the process by which a singleinfected cell produces many progeny virions that bud off and infectneighboring cells. The result is that a large area becomes rapidlyinfected, most of which was not initially infected by the original viralparticles. This is in contrast to vertical-type of infection in whichthe infectious agent spreads only through daughter progeny. Viralvectors can also be produced that are unable to spread laterally. Thischaracteristic can be useful if the desired purpose is to introduce aspecified gene into only a localized number of targeted cells.

Various methods can be used to introduce the expression vector of thepresent invention into stem cells. Such methods are generally describedin Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringsHarbor Laboratory, New York (1989, 1992), in Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.(1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich.(1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995),Vectors: A Survey of Molecular Cloning Vectors and Their Uses,Butterworths, Boston Mass. (1988) and Gilboa et at. [Biotechniques 4(6): 504-512, 1986] and include, for example, stable or transienttransfection, lipofection, electroporation and infection withrecombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and5,487,992 for positive-negative selection methods.

Introduction of nucleic acids by viral infection offers severaladvantages over other methods such as lipofection and electroporation,since higher transfection efficiency can be obtained due to theinfectious nature of viruses.

It will be appreciated that upregulation of MSF-A and/or HIF-1α can bealso effected by administration of MSF-A and/or HIF-1α-expressing cellsinto the individual.

MSF-A and/or HIF-1α-expressing cells can be any suitable cells, such ascardiac cells, bone marrow and lymphocyte cells which are derived fromthe individuals and are transfected ex vivo with one or two expressionvectors containing the polynucleotide(s) designed to express MSF-Aand/or HIF-1α as described hereinabove.

Administration of the MSF-A and/or HIF-1α—expressing cells of thepresent invention can be effected using any suitable route such asintravenous, intra peritoneal, intra kidney, intra gastrointestinaltrack, subcutaneous, transcutaneous, intramuscular, intracutaneous,intrathecal, epidural and rectal. According to presently preferredembodiments, the MSF-A and/or HIF-1α—expressing cells of the presentinvention are introduced to the individual using intravenous, intracardiac, intra gastrointestinal track and/or intra peritonealadministrations.

MSF-A and/or HIF-1α-expressing cells of the present invention can bederived from either autologous sources such as self bone marrow cells orfrom allogeneic sources such as bone marrow or other cells derived fromnon-autologous sources. Since non-autologous cells are likely to inducean immune reaction when administered to the body several approaches havebeen developed to reduce the likelihood of rejection of non-autologouscells. These include either suppressing the recipient immune system orencapsulating the non-autologous cells or tissues in immunoisolating,semipermeable membranes before transplantation.

Encapsulation techniques are generally classified as microencapsulation,involving small spherical vehicles and macroencapsulation, involvinglarger flat-sheet and hollow-fiber membranes (Uludag, H. et al.Technology of mammalian cell encapsulation. Adv Drug Deliv Rev. 2000;42: 29-64).

Methods of preparing microcapsules are known in the arts and include forexample those disclosed by Lu M Z, et al., Cell encapsulation withalginate and alpha-phenoxycinnamylidene-acetylated poly(allylamine).Biotechnol Bioeng. 2000, 70: 479-83, Chang T M and Prakash S. Proceduresfor microencapsulation of enzymes, cells and genetically engineeredmicroorganisms. Mol. Biotechnol. 2001, 17: 249-60, and Lu M Z, et al., Anovel cell encapsulation method using photosensitive poly(allylaminealpha-cyanocinnamylideneacetate). J. Microencapsul. 2000, 17: 245-51.

For example, microcapsules are prepared by complexing modified collagenwith a ter-polymer shell of 2-hydroxyethyl methylacrylate (HEMA),methacrylic acid (MAA) and methyl methacrylate (MMA), resulting in acapsule thickness of 2-5 μm. Such microcapsules can be furtherencapsulated with additional 2-5 μm ter-polymer shells in order toimpart a negatively charged smooth surface and to minimize plasmaprotein absorption (Chia, S. M. et al. Multi-layered microcapsules forcell encapsulation Biomaterials. 2002 23: 849-56).

Other microcapsules are based on alginate, a marine polysaccharide(Sambanis, A. Encapsulated islets in diabetes treatment. DiabetesTechnol. Ther. 2003, 5: 665-8) or its derivatives. For example,microcapsules can be prepared by the polyelectrolyte complexationbetween the polyanions sodium alginate and sodium cellulose sulphatewith the polycation poly(methylene-co-guanidine) hydrochloride in thepresence of calcium chloride.

It will be appreciated that cell encapsulation is improved when smallercapsules are used. Thus, the quality control, mechanical stability,diffusion properties, and in vitro activities of encapsulated cellsimproved when the capsule size was reduced from 1 mm to 400 μm (CanapleL. et al., Improving cell encapsulation through size control. J BiomaterSci Polym Ed. 2002; 13: 783-96). Moreover, nanoporous biocapsules withwell-controlled pore size as small as 7 nm, tailored surface chemistriesand precise microarchitectures were found to successfully immunoisolatemicroenvironments for cells (Williams D. Small is beautiful:microparticle and nanoparticle technology in medical devices. Med DeviceTechnol. 1999, 10: 6-9; Desai, T. A. Microfabrication technology forpancreatic cell encapsulation. Expert Opin Biol Ther. 2002, 2: 633-46).

An agent capable of upregulating the MSF-A or HIF-1α may also be anycompound which is capable of increasing the transcription and/ortranslation of an endogenous DNA or mRNA encoding the MSF-A or HIF-1αand thus increasing endogenous MSF-A or HIF-1α activity, respectively.

An agent capable of upregulating the MSF-A or HIF-1α may also be anexogenous polypeptide including at least a functional portion (asdescribed hereinabove) of the MSF-A or HIF-1α proteins.

Upregulation of MSF-A or HIF-1α can be also achieved by introducing atleast one MSF-A or HIF-1α substrate or inducer. Non-limiting examples ofsuch agents include PHD inhibitors, Capsaicin(8-methyl-N-Vanillyl-6nonenamide), DBM (dibenzoylmethane), CPX(ciclopirox olamine), Deferoxamine, Mersalyl, Chromium, CoCl₂ which areknown to induce HIF-1α expression and/or activity (Paul et al., 2004; J.Cell Physiol. 200: 20-30).

It will be appreciated that stabilization of the MSF-A-HIF1α proteincomplex can be also effected using a polypeptide capable of stabilizingthe MSF-A-HIF1α protein complex.

Each of the upregulating, stabilizing or downregulating agents describedhereinabove or the expression vector encoding MSF-A and/or HIF-1α orportions thereof can be administered to the individual per se or as partof a pharmaceutical composition which also includes a physiologicallyacceptable carrier. The purpose of a pharmaceutical composition is tofacilitate administration of the active ingredient to an organism.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Herein the term “active ingredient” refers to the upregulating,stabilizing or downregulating agent or the expression vector encodingMSF-A and/or HIF-1α which are accountable for the biological effect.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, various sugars and types of starch, cellulosederivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, especially transnasal, intestinal or parenteraldelivery, including intramuscular, subcutaneous and intramedullaryinjections as well as intrathecal, direct intraventricular, intravenous,inrtaperitoneal, intranasal, or intraocular injections.

Alternately, one may administer the pharmaceutical composition in alocal rather than systemic manner, for example, via injection of thepharmaceutical composition directly into a tissue region of a patient.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can beformulated readily by combining the active compounds withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the pharmaceutical composition to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions, and the like, for oral ingestion by a patient.Pharmacological preparations for oral use can be made using a solidexcipient, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethylcellulose, sodiumcarbomethylcellulose; and/or physiologically acceptable polymers such aspolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acidor a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for useaccording to the present invention are conveniently delivered in theform of an aerosol spray presentation from a pressurized pack or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in a dispenser may be formulated containing a powder mixof the compound and a suitable powder base such as lactose or starch.

The pharmaceutical composition described herein may be formulated forparenteral administration, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multidose containers with optionally, anadded preservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofthe active ingredients to allow for the preparation of highlyconcentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use.

The pharmaceutical composition of the present invention may also beformulated in rectal compositions such as suppositories or retentionenemas, using, e.g., conventional suppository bases such as cocoa butteror other glycerides.

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount ofactive ingredients (the upregulating, stabilizing or downregulatingagent or the expression vector encoding MSF-A and/or HIF-1α) effectiveto prevent, alleviate or ameliorate symptoms of a disorder (e.g., cancerand/or cancerous tumor or acute ischemia) or prolong the survival of thesubject being treated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromin vitro and cell culture assays. For example, a dose can be formulatedin animal models to achieve a desired concentration or titer. Suchinformation can be used to more accurately determine useful doses inhumans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient's condition(See e.g., Fingl, et al., 1975, in “The Pharmacological Basis ofTherapeutics”, Ch. 1 p. 1).

Dosage amount and interval may be adjusted individually to provideplasma levels of the active ingredient are sufficient to prevent cancerand/or cancerous tumor or acute ischemia (minimal effectiveconcentration, MEC). The MEC will vary for each preparation, but can beestimated from in vitro data. Dosages necessary to achieve the MEC willdepend on individual characteristics and route of administration.Detection assays can be used to determine plasma concentrations.

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks oruntil cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA approved kit, which may containone or more unit dosage forms containing the active ingredient. The packmay, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor administration. The pack or dispenser may also be accommodated by anotice associated with the container in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the compositions or human or veterinary administration. Suchnotice, for example, may be of labeling approved by the U.S. Food andDrug Administration for prescription drugs or of an approved productinsert. Compositions comprising a preparation of the inventionformulated in a compatible pharmaceutical carrier may also be prepared,placed in an appropriate container, and labeled for treatment of anindicated condition, as if further detailed above.

Thus, the teachings of the present invention can be used, for example,to treat individuals suffering from acute ischemia. Thus, an expressionvector (e.g., a viral vector) including a polynucleotide sequenceencoding the MSF-A and/or HIF-1α mRNA (SEQ ID NO:1 and/or 2,respectively) and the suitable promoter sequences to enable expressionin heart cells is introduced into the individual via intravenousadministration. Expression of such a vector in the heart is expected toupregulate and stabilize the MSF-A-HIF-1α protein complex in the heart,increase HIF-1α transcriptional activity on angiogenesis target genesand thus treat the acute ischemia. Dosage of such an expression vectorshould be calibrated using cell culture experiments and acute ischemiaanimal models. Success of treatment is preferably evaluated bydetermining the plasma levels of Troponin, CPK and other markers ofmyocardial acute ischemia and the individual general health status.

It will be appreciated, that if such a treatment is employed immediatelyfollowing the first signs of acute ischemia, i.e., during or immediatelyfollowing a heart attack, or stroke, it may prevent the complicationsassociated with such a condition.

The agents described hereinabove which are capable of increasing theMSF-A-dependent HIF-1α activity and/or stabilizing an MSF-A-HIF-1αprotein complex can be also used in various applications in whichupregulation of angiogenesis is desired.

Thus, according to another aspect of the present invention, there isprovided a method of inducing angiogenesis in a tissue. The method iseffected by contacting the tissue with an agent capable of upregulatingan MSF-A-dependent HIF-1α activity and/or stabilizing an MSF-A-HIF-1αprotein complex to thereby induce angiogenesis in the tissue.

As used herein the term “angiogenesis” refers to the formation of newblood vessels, usually by sprouting from pre-existing blood vessels.

The term “tissue” refers to aggregate of cells having a similarstructure and function and including blood vessels. Examples include,but are not limited to, brain tissue, retina, skin tissue, hepatictissue, pancreatic tissue, bone, cartilage, connective tissue, bloodtissue, muscle tissue, cardiac tissue brain tissue, vascular tissue,renal tissue, pulmonary tissue, gonadal tissue, hematopoietic tissue andfat tissue.

The tissue according to this aspect of the present invention can be partof an organism or individual (i.e., for in vivo angiogenesis), can betaken out of the organism (e.g., for ex vivo tissue repair) or can beformed ex vivo from cells derived from the organism on a scaffold or amatrix selected suitable for tissue formation.

For example, the agents according to this aspect of the presentinvention which are capable of upregulating an MSF-A-dependent HIF-1αactivity and/or stabilizing an MSF-A-HIF-1α protein complex can be usedto induce angiogenesis of a tissue. Briefly, any of the MSF-A and/orHIF-1α upregulating agents of the present invention (e.g., thepolynucleotide expressing MSF-A or HIF-1α, the MSF-A or HIF-1αpolypeptide or peptide, cells expressing MSF-A or HIF-1α, the compoundincreasing MSF-A or HIF-1α transcription, translation or stabilityand/or the MSF-A or HIF-1α substrate) can be provided to a tissue tothereby activate HIF-1α activity and induce angiogenesis.

Thus, the method and agents (which are capable of increasing theMSF-A-dependent HIF-1α activity) according to this aspect of the presentinvention can be used in vitro, to form a tissue model, ex vivo, fortissue regeneration and/or repair and in vivo for tissue regenerationand/or repair in various clinical conditions such as infarcted heart,brain lesion, spinal cord injury, ischemia and the like.

According to preferred embodiments of this aspect of the presentinvention, the MSF-A and/or HIF-1α upregulating agents can be attachedto, added to or impregnated within a scaffold designed to enable cellgrowth, angiogenesis and tissue formation. Such a scaffold can be anysynthetic or biodegradable scaffold known in the arts. Non-limitingexamples of scaffolds which can be used to induce angiogenesis alongwith the agents of the present invention include the bioengineeredpolyglycolic acid cloth (PGAC) described in Fukuhara S., et al. (Circ.J. 69:850-7, 2005), hyaluronic acid (HA) hydrogels (Hou S, et al., 2005,J. Neurosci. Methods. June 21; Epub ahead of print), fibrin gel (Royce SM, et al., 2004, J. Biomater. Sci. Polym. Ed. 15(10): 1327-36) and thelike.

The present invention also envisages identification of other anti canceragents which are capable of preventing the formation of or dissociatingthe MSF-A-HIF-1α protein complex and as such may be used as anti cancerdrugs.

Thus, according to another aspect of the present invention there isprovided a method of identifying putative anti cancer agents.

As used herein, the phrase “anti cancer agents” refers to chemicals,antibodies, aptamers, peptides and the like which can be used to treatand prevent the growth of cancerous cells or cancerous tumors.

The method is effected by identifying agents which are capable ofdownregulating an MSF-A dependent HIF-1α activity, preventing theformation of or dissociating the MSF-A-HIF-1α protein complex to therebyidentify the putative anti cancer agents.

As is shown in Example 1 of the Examples section which follows anMSF-A-HIF-1α protein complex used for identifying such agents (i.e., thepre-established complex) can be formed in vitro by co-transfection ofcells with expression vectors containing the MSF-A (SEQ ID NO:1) andHIF-1α (SEQ ID NO:2) coding sequences.

Alternatively, peptides, which encompass the interaction site of eitherof the proteins may be used to generate the MSF-A-HIF-1α protein complexof this aspect of the present invention. For example, a peptide which isderived from the N-terminal part of the MSF-A protein (i.e., amino acids1-25 as set forth in SEQ ID NO:3) along with additional amino acidsequences (as needed) can be used to form the pre-established complex.

Combinatorial chemical, nucleic acid or peptide libraries may be used toscreen a plurality of agents.

Screening according to this aspect of the present invention may beeffected by contacting the agents with the pre-established complexdescribed hereinabove or with either an MSF-A or an HIF-1α. The MSF-A orthe HIF-1α proteins are preferably bound to a solid support to monitorbinding of the agent to the MSF-A or the HIF-1α proteins or to monitordissociation of the pre-established complex, respectively. The solidsupport may be any material known to those of ordinary skill in the artto which a specific antibody which can recognize the MSF-A or the HIF-1αproteins may be attached, such as a test well in a microtiter plate, anitrocellulose filter or another suitable membrane. Alternatively, thesupport may be a bead or disc, such as glass, fiberglass, latex or aplastic such as polystyrene or polyvinylchloride. Molecular immobilizionon a solid support is effected using a variety of techniques known tothose in the art.

A number of methods are known in the art for determining intermolecularinteractions. Examples include, but are not limited to, ELISA, Biacore,Pull-down assay, immunoprecipitation and the like (see references in theExamples section which follows).

A competitive assay in which at least one of the assay component islabeled may also be employed. Labeling methods and tags are described inthe references incorporated to the Examples section which follows.

It will be appreciated that when utilized along with automatedequipment, the above-described method can be used to screen multipleagents both rapidly and easily.

Agents identified using the above-described methodology can be furtherqualified by functional assays, such as by inhibiting thetranscriptional activity of a reporter gene (e.g., luciferase) asdescribed in Example 2 of the Examples section which follows.

As is shown in FIGS. 6 a-d, an immunoprecipitation experiment followedby an immunoblotting experiment demonstrated that an N-terminaltruncated form of MSF-A is unable to form a protein complex with HIF-1α.

Thus, the present invention further provides a method of determining ifa molecule is capable of preventing the formation of and/or dissociatingan MSF-A-HIF-1α protein complex.

The method is effected by incubating the MSF-A-HIF-1α protein complex orcells harboring the MSF-A-HIF-1α protein complex with the molecule (forexample, any one of the peptides set forth by SEQ ID Nos:12-4193 and4213) and determining the presence, absence or level (amount ofcomplexed vs. uncomplexed proteins) of the MSF-A-HIF-1α protein complexfollowing such incubation. It will be appreciated that absence of theMSF-A-HIF-1α protein complex is indicative of the capacity of themolecule to prevent the formation of and/or dissociate the MSF-A-HIF-1αprotein complex.

As is shown in the Examples section which follows, the incubation timeused by the present invention to detect the presence or absence of theMSF-A-HIF-1α protein complex was in most cases 24-48 hours followingtransfection, or 24 hours following subjecting the cells to hypoxia ornormoxia.

Thus, according to preferred embodiments incubating is effected for atime period selected from the range of 1-48 hours, more preferably, fora time period of 1-24 hours, most preferably, 1-12 hours.

The term “determining” as used herein with regard to the presence orabsence of the protein complex, refers to the detection, identificationor isolation of the protein complex (i.e., via immunoprecipitation andaffinity columns) and the determination of the presence of both proteins(i.e., MSF-A and HIF-1α) within such a protein complex using e.g., animmunological detection method as is shown in FIGS. 3 a-f and theExamples section which follows.

Immunological detection methods: The immunological detection methodsused in context of the present invention are fully explained in, forexample, “Using Antibodies: A Laboratory Manual” [Ed Harlow, David Laneeds., Cold Spring Harbor Laboratory Press (1999)] and those familiarwith the art will be capable of implementing the various techniquessummarized hereinbelow as part of the present invention. All of theimmunological techniques require antibodies specific to both MSF-A andHIF-1α proteins. Such antibodies can be obtained from any commercialsupplier of molecular biology reagents such as Gibco-InvitrogenCorporation (Grand Island, N.Y. USA), Sigma (St. Louis Mo., USA), SantaCruz (Biotechnology, Inc., Santa Cruz, Calif., USA), Roche(Indianapolis, Ind., USA) and/or by using the MSF-A antibody of thepresent invention Immunological detection methods suited for use as partof the present invention include, but are not limited to,radio-immunoassay (RIA), enzyme linked immunosorbent assay (ELISA),western blot, immunohistochemical analysis, and fluorescence activatedcell sorting (FACS).

Radio-immunoassay (RIA): In one version, this method involvesprecipitation of the desired substrate, HIF-1α or MSF-A in this case andin the methods detailed hereinbelow, with a specific antibody andradiolabelled antibody binding protein (e.g., protein A labeled withI¹²⁵) immobilized on a precipitable carrier such as agarose beads. Thenumber of counts in the precipitated pellet is proportional to theamount of substrate.

In an alternate version of the RIA, a labeled substrate and anunlabelled antibody binding protein are employed. A sample containing anunknown amount of substrate is added in varying amounts. The decrease inprecipitated counts from the labeled substrate is proportional to theamount of substrate in the added sample.

Enzyme linked immunosorbent assay (ELISA): This method involves fixationof a sample (e.g., fixed cells or a proteinaceous solution) containing aprotein substrate to a surface such as a well of a microtiter plate. Asubstrate specific antibody coupled to an enzyme is applied and allowedto bind to the substrate. Presence of the antibody is then detected andquantitated by a colorimetric reaction employing the enzyme coupled tothe antibody. Enzymes commonly employed in this method includehorseradish peroxidase and alkaline phosphatase. If well calibrated andwithin the linear range of response, the amount of substrate present inthe sample is proportional to the amount of color produced. A substratestandard is generally employed to improve quantitative accuracy.

Western blot: This method involves separation of a substrate from otherprotein by means of an acrylamide gel followed by transfer of thesubstrate to a membrane (e.g., nylon or PVDF). Presence of the substrateis then detected by antibodies specific to the substrate, which are inturn detected by antibody binding reagents. Antibody binding reagentsmay be, for example, protein A, or other antibodies. Antibody bindingreagents may be radiolabelled or enzyme linked as described hereinabove.Detection may be by autoradiography, colorimetric reaction orchemiluminescence. This method allows both quantitation of an amount ofsubstrate and determination of its identity by a relative position onthe membrane which is indicative of a migration distance in theacrylamide gel during electrophoresis.

Immunohistochemical analysis: This method involves detection of asubstrate in situ in fixed cells by substrate specific antibodies. Thesubstrate specific antibodies may be enzyme linked or linked tofluorophores. Detection is by microscopy and subjective evaluation. Ifenzyme linked antibodies are employed, a colorimetric reaction may berequired. It will be appreciated that the presence of the MSF-A-HIF-1αcomplex can be detected in cells by double-labeling immunofluorescenceas described in Example 3 of the Examples section which follows.

Fluorescence activated cell sorting (FACS): This method involvesdetection of a substrate in situ in cells by substrate specificantibodies. The substrate specific antibodies are linked tofluorophores. Detection is by means of a cell sorting machine whichreads the wavelength of light emitted from each cell as it passesthrough a light beam. This method may employ two or more antibodiessimultaneously.

According to preferred embodiments of the present inventiondetermination of the presence or absence of the MSF-A-HIF-1α proteincomplex is effected by sequentially and/or simultaneously exposing theprotein complex or cells expressing the protein complex to an anti-MSF-Aand anti-HIF-1α antibodies.

As used herein the term “sequentially” refers the use of one antibody inone immunological detection method (i.e., immunoprecipitation oraffinity column) and the use of the second antibody in the otherimmunological detection method (i.e., Western Blot, Eliza, FACS and thelike). See for further details the methodology described in the Examplessection which follows.

The term “simultaneously” as used herein refers to the use of bothantibodies by the same time, using for example, doubleimmunohistochemistry (see FIGS. 12 a-c and Example 3 of the Examplessection which follows).

As is mentioned before, the association of MSF-A with the HIF-1α proteinupregulates HIF-1α transcriptional activity (see Example 2 of theExamples section which follows). In addition, HIF-1α over-expression isassociated with a failure of anti cancer therapy [Escuin, 2004 (Supra)].

Thus, the present invention also contemplates a method of determiningthe prognosis of an individual having cancer.

The method is effected by determining the presence or absence of anMSF-A-HIF-1α protein complex in cancerous cells derived from theindividual, wherein the presence of such a protein complex is indicativeof poor prognosis of the individual.

As used herein “prognosis” refers to the probable outcome or course of adisease; the chance of recovery. Thus, individuals with poor prognosishave less chances of recovery than individuals with good prognosis.

It will be appreciated that individuals in which the MSF-A-HIF-1αprotein complex is detected have relatively poor prognosis as comparedwith individuals lacking such a protein complex.

As used herein the term “about” refers to ±10%.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., Ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(Eds.) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., Ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., Ed. (1994); Stites et al.(Eds.), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (Eds.), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., Ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,Eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., Eds. (1984); “Animal Cell Culture” Freshney, R. I., Ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference as if fully set forth herein. Other general references areprovided throughout this document. The procedures therein are believedto be well known in the art and are provided for the convenience of thereader. All the information contained therein is incorporated herein byreference.

General Materials and Experimental Methods

Metabolic labeling of PC-3 cells—Metabolic labeling of PC-3 cells wasperformed using [³⁵S] methionine (ICN Biomedicals, Inc., CA) essentiallyas described in Mabjeesh, N. J. et al. (Geldanamycin induces degradationof hypoxia-inducible factor 1 alpha protein via the proteasome pathwayin prostate cancer cells. Cancer Res., 62: 2478-2482, 2002).

Preparation of whole cell lysates—Cells were washed twice with ice-coldPBS, harvested whole cell extracts (WCE) were prepared by lysing thecells with 100 mM potassium phosphate (pH 7.8) and 0.2% Triton X-100supplemented with protease and phosphatase inhibitors as describedelsewhere (Mabjeesh N J et al. 2ME2 inhibits tumor growth andangiogenesis by disrupting microtubules and dysregulating HIF. CancerCell 2003; 3: 363-375).

Immunoblotting—Proteins (30-60 μg/lane) from WCEs were resolved by 7.5%SDS-PAGE, electro-blotted to nitrocellulose membranes and incubated withthe indicated primary antibodies, followed by horseradishperoxidase-conjugated secondary antiserum (Amersham Biosciences,Piscataway, N.J.). Immunoreactivity was visualized by Amersham enhancedchemiluminescence reagent (Amersham Biosciences, Piscataway, N.J.). Forsequential blotting with additional antibodies, the membranes werestripped from the first primary antibody using a restore Western blotstripping buffer (Pierce, Rockford, Ill.) and were re-probed using thesecond primary antibody as indicated.

Immunoprecipitation—Cells were washed twice with ice-cold PBS, lysed in20 mM Na-HEPES, pH 7.5, 0.5% Nonidet P-40, 0.1M NaCl, 2 mM EDTA, 10%Glycerol and 2 mM DTT supplemented with protease and phosphataseinhibitors, and were subjected to immunoprecipitation using anti-HIF-1αantibody and protein G-agarose beads (Pierce, Ill.) according to themanufacturer's instructions.

Construction of the p3xFlag-HIF-1α vector—HIF-1α cDNA (GenBank accessionNo. NM_(—)001530; SEQ ID NO:2) was subcloned at NotI/XbaI sites of thep3XFLAG-myc-CMV-25 vector (Sigma-Aldrich Corp., St Louis, Mo., USA) toprovide Flag-tagged HIF-1α at its N-terminal using the following PCRprimers: forward 5′-acgtgeggccgcgatggagggcgccggcggcgcgaacg-3′ (SEQ IDNO:8) and reverse 5′-cagttctagattatcagttaacttgatccaaagctctgag-3′ (SEQ IDNO:9).

Construction of the pcDNA3.1-HIF-1α expression vector—HIF-1α cDNA wascut from the p3xFlag-HIF-1α vector at the NotI/XbaI sites and pasted tothe pcDNA3.1(+) expression vector (Invitrogen Life Technologies,Carlsbad, Calif.) to obtain the untagged HIF-1α wild-type.

Construction of the MSF-A expression vector (p3xFLAG-MSF-A)—Total RNAprepared from PC-3 cells was subjected to an RT-PCR reaction using theforward [5′-GACTAAGCTTATGAAGAAGTCTTACTCAGGAGGCACGCGG-ACC-3′ (SEQ IDNO:4)] and reverse[5′-ACGTTCTAGATTACTA-CATCTCTGGGGCTTCTGGCTCCTTCTCCTCC-3′ (SEQ ID NO:5)]PCR primers designed according to MSF-A cDNA sequence (GenBank AccessionNo AF189713, SEQ ID NO:1). The resultant MSF-A cDNA was subcloned intothe p3xFLAG-myc-CMV-25 (Sigma-Aldrich Corp., St Louis, Mo., USA) atHindIII/XbaI sites to provide the FLAG sequence at the amino terminal ofthe recombinant protein. The sequence of the cloned MSF-A vector wasvalidated by sequence analysis.

Construction of ΔN-MSF-A expression vector (p3xFlag-ΔN-MSF-A)—Thep3xFLAG-MSF-A vector was subjected to PCR using the forward[5′-GACAAGCTTGCCTTGAAAAGATCTTTTGAGGTC-3′ (SEQ ID NO:6)] and reverse (SEQID NO:5)] PCR primers and the resultant N-terminal truncated MSF-A cDNA(ΔN-MSF-A; deletion of the first 25 amino acids, SEQ ID NO:7) wassubcloned into the p3xFLAG-myc-CMV-25 at the HindIII/XbaI sites.

Construction of ΔG-MSF-A expression vector (p3XFlag-ΔG-MSF-A)—The MSF-Amutant lacking the GTP binding domain [ΔG; deletion of amino acidsGQSGLGKS (SEQ ID NO:4198) which correspond to amino acids 305-312 as setforth by SEQ ID NO:3] was generated by two PCR reactions using thevector containing the wild-type MSF-A (p3xFLAG-MSF-A) as a template. Inthe first PCR reaction, the forward5′-GACTAAGCTTATGAAGAAGTCTTACTCAGGAGGCACGCGGACC-3′ (SEQ ID NO:4) primercorresponding to the N-terminal and an internal reverse primer5′-TGGATTTGAAGAGGGTGTTGATTAAGGTGACCACCATGATGTTGAACTCG AAGCCC-3′ (SEQ IDNO:4199) lacking 24 nucleotides corresponding to the GTP bindingsequence (SEQ ID NO:4198). In the second reaction, the antisensesequence of SEQ ID NO:4199 was used as a forward internal primer5′-GGGCTTCGAGTTCAACATCATGGTGGTCACCTTAATCAACACCCTCTTCAA ATCCA-3′ (SEQ IDNO:4216) and the reverse primer was5′-ACGTTCTAGATTACTACATCTCTGGGGCTTCTGGCTCCTTCTCCTCC-3′ (SEQ ID NO:5)corresponding to the C-terminus of MSF-A. The overlapping two PCRproducts were used as a template to obtain the full-length of ΔG mutantof MSF-A using the external primers (SEQ ID NOs:4 and 5). The productwas subcloned into p3XFlag-myc-CMV-25 vector at the HindIII and XbaIsites.

Stable transfection—PC-3 cell were seeded at 50% confluence in 100mm-diameter dishes and grown in complete medium for overnight.Transfections were carried out with p3XFlag-CMV-myc-25 which includes aneomycin-resistance gene. Cells were transiently transfected with eitherthe empty vector (EV; p3xFLAG-myc-CMV-25) or the vector carrying MSF-A(p3xFLAG-MSF-A). After 48 hours, the medium was replaced with freshmedium supplemented with 1 gr/ml of neomycin (G418; Sigma-Aldrich Co.,Saint Louis, Mo.) and was changed every 3 days. Two to three weekslater, neomycin-resistant colonies were isolated and grown in mediumsupplemented with 500 mg/ml of neomycin. Cells were screened forFlag-MSF-A expression by Western blotting. To avoid clonal variation ofthe stably-transfected cells, 30 neomycin resistant clones of PC-3stably transfected with the empty vector were pooled together anddesignated “PC-3-Neo” and 30 resistant clones stably-transfected withMSF-A were pooled together and designated as “PC-3-MSF-A”.

Reporter gene assay and luminescence measurements—HRE-dependentluciferase activity was performed using the pBI-GL construct (pBI-GLV6L) containing six tandem copies of the VEGF hypoxia response elementas previously described (Mabjeesh et al., 2002; Post and Van Meir,2001). Cells were grown in E-well plates and then transientlytransfected in triplicate with a total of 1 μg DNA including 0.1 μg ofthe reporter plasmid, pBI-GL V6L. Duplicate sets of transfectedcell-culture plates were then separated and incubated for 16 hours undereither normoxic or hypoxic conditions. Luciferase enzymatic activity wasmeasured using the commercial kit TROPIX (Bedford, Mass.) in the Galaxyluminometer (BMG Labtechnologies LUMIstar) following the manufacturer'sinstructions. Arbitrary Luciferase activity units were normalized to theamount of protein in each assay point.

Isolation and analysis of RNA—Total RNA was extracted from cells byusing RNeasy Mini Kit (Qiagen Inc., Valencia, Calif.) according to themanufacturer's protocol. Total RNA from xenograft-derived tumors, whichwere frozen in liquid N₂ immediately after dissection, was preparedusing TRI REAGENT (Sigma-Aldrich Co., Saint Louis, Mo.) following themanufacturer's instructions. One μg total RNA was reverse transcribedinto cDNA using Reverse-iT 1^(st) Strand Synthesis Kit (ABgene, Epsom,United Kingdom) using anchored oligo dT as first strand primer. PCR wasperformed in 25 μl reaction mixture using ReddyMix PCR master mix(ABgene, Epsom, United Kingdom) with 0.3 μM of each primer and 50 ngtemplate. Semi-quantitative RT-PCR was performed using β-actin as aninternal control to normalize gene expression for the PCR templates. PCRcycle number was optimized for each primer set. Representative sampleswere run at different cycle numbers and the optimal cycle number wasselected in the region of linearity between cycle number and PCR productintensity. Sequences of the PCR primers, number of cycles, annealingtemperature and product size for each gene were as follow:

VEGF (GenBank Accession No. NM_(—)00376): forward primer5′-tcttcaagccatcctgtgtg-3′ (SEQ ID NO:4200), reverse primer5′-tctctcctatgtgctggcct-3′ (SEQ ID NO:4201), 22 cycles, annealingtemperature 57° C., 144 by PCR product.

HIF-1α (GenBank Accession No. NM_(—)001530): forward primer5′-ggacaagtcaccacaggaca-3′ (SEQ ID NO:4202), reverse primer5′-gggagaaaatcaagtcgtgc-3′ (SEQ ID NO:4203), 25 cycles, annealingtemperature 56° C., 169 by PCR product.

β-actin (GenBank Accession No. NM_(—)001101): forward primer5′-ctcctgagcgcaagtactcc-3′ (SEQ ID NO:4204), reverse primer5′-ctgcttgctgatccacatctg-3′ (SEQ ID NO:4205), 17 cycles, annealingtemperature 55° C., 86 by PCR product.

ET-1 (GenBank Accession No. NM_(—)001955): forward primer5′-ccatgagaaacagcgtcaaa-3′ (SEQ ID NO:4206), reverse primer5′-agtcaggaaccagcagagga-3′ (SEQ ID NO:4207), 22 cycles, annealingtemperature 57° C., 213 by PCR product.

CA-IX (GenBank Accession No. NM_(—)001216): forward primer5′-caaagaaggggatgaccaga-3′ (SEQ ID NO:4208), reverse primer5′-gaagtcagagggcaggagtg-3′ (SEQ ID NO:4209), 26 cycles, annealingtemperature 57° C., 568 by PCR product.

Glut-1 (GenBank Accession No. NM_(—)006516): forward primer5′-gggcatgtgcttccagtatgt-3′ (SEQ ID NO:4210), reverse primer5′-accaggagcacagtgaagat-3′ (SEQ ID NO:4211), 33 cycles, annealingtemperature 57° C., 72 by PCR product.

MSF-A (GenBank Accession No. AF189713): forward primer (SEQ ID NO:4),reverse primer (SEQ ID NO:5 or 4199), 22 cycles, annealing temperature55° C., PCR products of 1761 by or 940 bp, respectively.

The PCR products were visualized by UV illumination followingelectrophoresis through 2% agarose containing 0.5 μg/ml ethidium bromideat 50 V for 1 hour.

HIF-1α protein stability assays—Cells were plated into 6-well plates andgrown to 70% confluence. The cells were subjected to eithercycloheximide (CHX) treatment or to metabolic labeling and pulse chaseassay. CHX was added to the cells at a final concentration of 10 μg/mlfor the indicated time in the FIGS. 16 a-c. Subsequently, the cells werewashed and harvested for immunoblotting. Cells were metabolicallylabeled as previously described (Mabjeesh, N. J., et al., 2002, CancerRes 62: 2478-2482). Briefly, the medium was changed to methionine- andcysteine-free as well as serum-free RPMI 1640 medium for 2 hours,following which the cells were labeled by incubation with methionine-and cysteine-free medium containing ³⁵S-methionine (Amersham BiosciencesCorp., Piscataway, N.J.) at a final concentration of 100 μCi/well at 37°C. for 1.5 hours. Subsequently, the radioactive medium was removed andcells were re-cultured in complete medium for the indicated times. Thecells were washed twice with ice-cold PBS, lysed, and subjected toimmunoprecipitation using the anti-HIF-1α antibody Immunoprecipitateswere analyzed on SDS-PAGE and visualized by autoradiography.

Immunohistochemistry with anti-CD34 and anti-Ki67—Forimmunohistochemical staining paraffin-embedded tissue was sectioned at 3μm and mounted on Superfrost/plus slides (Menzel-Glaser, Braunschweig,Germany). Sections were processed by a labeled-(strept)-avidin-biotin(LAB-SA) method using a Histostain Plus Kit (Zymed, San Francisco,Calif.) and following exactly the manufacturer's instructions. Sectionswere pre-treated for 12 minutes at 97° C. with the Target Retrievalbuffer (Zymed, San Francisco, Calif.) at pH 6.0 using atemperature-controlled microwave (H2800 model processor, Energy BeamSciences Inc., Apawa, Mass.). The sections were treated with 3% H₂O₂ for5 minutes, followed by 10 minutes incubation with the universal blocker,Cas-Block (Zymed, San Francisco, Calif.). After blocking, sections wereincubated for 30 minutes at room temperature with anti-CD34 (at a 1:50dilution) or anti-Ki67 (at a 1:25 dilution). Slides were then washedwith the TNT wash buffer (NEN, CITY, COUNTRY) and incubated for 30minutes with species-specific biotinylated secondary antibody (1:200dilution, Vector Laboratories, Burlingame, Calif.). A biotinylatedsecondary antibody was applied for 10 minutes, followed by a 10-minuteincubation with peroxidase conjugated streptavidin (HRP-SA). The slideswere thoroughly washed after each incubation using the Optimamax washbuffer (Biogenex, San Roman, Calif.). The immunoreaction was visualizedby an HRP-based chromogen (Substrate System) including diamino-benzidinebrown chromogene (Liquid DAB Substrate Kit, Zymed). The sections werethen counterstained with Mayen's hematoxylin dehydrated in ascendingethanol concentration, cleared in xylene and mounted for microscopicexamination. For negative controls, the exact procedure was done withthe omission of either the primary or the secondary antibody. Forquantitative analysis of Ki67 and CD34 staining, positive staining cellsand microvessels were counted and their density expressed as the numberof positive cells per total number of cells or capillaries per totalsection area excluding necrotic areas, respectively.

Production of sequence-directed antibodies against MSF-A protein—Apeptide of 15 amino acids corresponding to the amino terminal part ofMSF-A protein (amino acids 3-17; KSYSGGTRTSSGRLR; SEQ ID NO:4212) wassynthesized, conjugated to a carrier protein and injected into rabbits(Convance Research Products Inc., Denver, Pa.). The sequence of thepeptide was selected from the region that is not homologous to any othermember within the overall septin family. The sera drawn from the rabbitswere tested for MSF-A immunoreactivity using IP and Western blotting.

Immunofluorescence and confocal microscopy—Exponentially growing cellswere plated on 12-mm glass coverslips (Fisher Scientific, Pittsburgh,Pa.) into 24-well plates and cells were allowed to attach overnight. Thefollowing day, cells were subjected to hypoxia for 16 hours. Cells werefixed for 10 minutes at room temperature with the PHEMO buffer (PIPES0.068 M, HEPES 0.025 M, EGTA 0.015 M, MgCl₂ 0.003 M, 10% DMSO, pH 6.8)containing 3.7% formaldehyde, 0.05% glutaraldehyde, 0.5% Triton X-100.Coverslips were blocked for 10 minutes in 10% goat serum/PBS andprocessed for double-labeling immunofluorescence with monoclonal mouseanti-HIF-1α, and polyclonal rabbit anti-MSF-A antibodies. The secondaryantibodies were Alexa Fluor 488 goat anti-mouse antibody and RhodamineRed-X goat anti-rabbit antibodies. Coverslips were then mounted ontoglass slides and examined with a Zeiss axioplasm laser scanning confocalmicroscope using a Zeiss×100 1.3 oil-immersion objective.

In vitro Proliferation Assays

Cell proliferation assay with XTT reagent—For cell proliferation assay,PC-3-Neo and PC-3-MSF-A cells were seeded in 96 well-plates (1000cells/well in 200 μl) using a 3-bis-(2-methoxy-4-nitro-5sulfenyl)-(2H)-tetrazolium-5-carboxanilide (XTT) kit (BiologicalIndustries Ltd. Kibbutz Beit Haemek, Israel). Wells filled with mediaserved as a control. On the next day, the cells were cultured eitherunder normoxic or hypoxic conditions. XTT reagent was added at days 0,2, 4 and 6 following the manufacturer's instructions. The absorbance ofthe samples was measured using a microplate reader at a wavelength of450 nm using Elisa reader 680 (Bio-rad, Hercules, Calif.). Allexperiments were performed in triplicate.

Plating Efficiency assay—PC-3-Neo and PC-3-MSF-A cells were cultured in100 mm-diameter plate (1000 cells/plate) and incubated to allow colonyformation. The cultures were monitored on a daily basis and whencolonies were visible (approximately after 2 weeks) cells were fixed andstained with 90% EtOH, 5% Acetic acid, 0.01% Coomassie brilliant blue(Sigma-Aldrich, Saint Louis, Mo.). Plating was done in triplicates.Colonies containing ≧20 cells were counted. Plating efficiency (%) wascalculated as number of colonies formed/number of cells plated×100.

Soft Agar Foci assay—The assay tests the anchorage-independent growth ofthe cell in soft agar. Suspensions of PC-3-Neo or PC-3-MSF-A cells in0.22% soft agar were poured on 35 mm-diameter plate (5000 cells/plate)pre-cast with 0.5% soft agar and were cultured for 4 weeks. Colonies(≧20 cells) were counted.

Tumor models and immunohistochemistry—PC-3-Neo or PC-3-MSF-A cells(3×10⁶) were subcutaneously (s.c.) injected into the right hinds ofCD1/nude mice. All procedures were performed in compliance with the TelAviv Sourasky Medical Center Animal Care and Use Committee and NIHguidelines. Animals were monitored for tumor development twice a week.Tumor parameters were measured with calipers, and tumor volume wascalculated according to the formula: tumor volume=width²×length×0.52.After 6 weeks, animals were sacrificed and tumors were excised asquickly as possible, weighed and cut into 2 pieces. One piece of tumorwas fixed with 4% buffered formalin for 24 hours, embedded in Paraplast(Oxford Labware, St. Louis, Mo., USA) until immunohistochemical stainingand the second piece of the tumor was immediately frozen in liquid N₂and kept at −80° C. for RNA analysis.

Tumor array and dot blot analysis—An MSF-A cDNA [HindIII/EcoRI; SEQ IDNO:4214 (nucleotides 1-721 of SEQ ID NO:1)] was labeled with α³²P-dCTP(Qiagen Inc., Valencia, Calif.) and used to probe the Human MatchedTumor/Normal Expression Array from Clontech Cat. #7840-1 (Mountain View,Calif.) under high stringency conditions following the instructions ofthe manufacturer. Washed filters were exposed to autoradiographic films.For normalization, the membrane was stripped and re-probed with alabeled β-actin probe (Ambion, Austin, Tex., USA). Dots densities wereanalyzed using densitometry and each MSF-A dot was normalized to itscorresponding β-actin dot. The ratio between tumoral and normalexpression of each pair was then calculated.

Data analysis—Experiments presented in all Figures of the presentinvention are representative of three or more different repetitions.Quantification of band densities was performed using the public domainNIH Image (version 1.61). Statistical analysis was performed using aone-way ANOVA test (p<0.05 was considered statistically significant).

Example 1 MSF-A Associates with HIF-1 Alpha In Vitro and In Vivo

Prostate cancer cells (PC-3) express increased levels of HIF-1α proteinunder normoxic conditions (Zhong H et al. Increased expression ofhypoxia inducible factor-1alpha in rat and human prostate cancer. CancerRes 1998; 58: 5280-5284). To identify proteins, which regulate HIF-1transcriptional activity under aerobic/normoxic conditions, PC-3 cellswere used in a set of immunoprecipitation (IP) and immunoblotting (IB)analyses, as follows.

Experimental Results

MSF-A co-immunoprecipitated with HIF-1α—PC-3 cells were metabolicallylabeled with [³⁵S]-methionine and whole cell lysates were subjected toimmunoprecipitation using a purified mouse monoclonal anti-HIF-1αantibody (BD Transduction Laboratories, Lexington, Ky.). Theimmunoprecipitated proteins were analyzed on an SDS-PAGE and werevisualized by autoradiography. As is shown in FIG. 1, several proteinbands were detected in the immunoprecipitate protein mixture. Of theseproteins, a 70 kDa protein, displayed a strong band signal (FIG. 1),suggesting strong association with the HIF-1α protein. Anotherrelatively strong band, of 120 kDa, corresponded to the HIF-1α protein.To determine the identity of the 70-kDa protein, a larger scale of anon-radioactive PC-3 cell preparation was subjected to silver staining,following which the 70-kDa band was eluted and its amino acid sequencewas analyzed using MALDI-TOF-MS. The protein was identified as amyeloid/lymphoid leukemia septin-like fusion protein A (MSF-A, GenBankAccession No. AAF23374, SEQ ID NO:3). This septin-like protein was firstidentified as part of a fusion protein with MLL in a therapy-inducedacute myeloid leukemia patient [Osaka M, Rowley J D, Zeleznik-Le N J.MSF (MLL septin-like fusion), a fusion partner gene of MLL, in atherapy-related acute myeloid leukemia with a t(11;17)(q23;q25). ProcNatl Acad Sci USA 1999; 96: 6428-6433]. The exact function and cellularlocalization of MSF-A protein have not been elucidated. Recent studiessuggested novel functions for septins in vesicle trafficking,cytokenesis and oncogenesis (Kartmann B, Roth D. Novel roles formammalian septins: from vesicle trafficking to oncogenesis. J Cell Sci2001; 114: 839-844).

Confirmation of MSF-A/HIF-1α interaction—Since the MSF-A protein has notbeen previously characterized, antibodies capable of recognizing thisprotein have not been available prior to the present study. In order toconfirm the interaction between MSF-A and HIF-1α, an in vitro systemcomprising of co-transfected cells was established. In a preliminaryexperiment, PC-3 or HEK 293 cells were transiently transfected with thep3xFlag-MSF-A expression vector and cell lysates of transfected cellswere subjected to Western Blot analysis using an anti-Flag antibody(Sigma-Aldrich Corp., St Louis, Mo., USA). As is shown in FIG. 2, atime-dependent expression of the recombinant 70-kDa FLAG-tagged proteinwas observed.

To confirm the interaction between the HIF-1α and the MSF-A proteins,HEK 293 cells were transiently co-transfected with the p3xFlag-MSF-A andthe pcDNA3.1-HIF-1α vectors, and were grown for 24 hours under normoxicconditions (i.e., conditions of 20% oxygen), following which whole cellextracts (WCE) were subjected to co-immunoprecipitation (IP) usinganti-FLAG or anti-HIF-1α antibodies. Following IP, the proteins weresubjected to SDS-PAGE and immunoblotting (IB) using the counterpartantibody. As is shown in FIGS. 3 a-d, following transfection with bothexpression vectors (i.e., pcDNA3.1-HIF-1α and p3xFlag-MSF-A)immunoprecipitates generated using either the anti-HIF-1α antibody orthe anti-Flag antibody included the reciprocal proteins, i.e., theFlag-MSF-A or HIF-1α proteins, respectively. These findings clearlydemonstrate a strong association between the two recombinant proteins(i.e., MSF-A and HIF-1α) in vitro. On the other hand, as is furthershown in FIGS. 3 e-f, the Flag antibody (which represents the MSF-Aprotein) failed to co-immunoprecipitate HIF-1β protein.

To further confirm the HIF-1α/MSF-A interaction, HEK 293 cells weretransiently co-transfected with both the p3xFlag-MSF-A andp3xFlag-HIF-1α vectors. Twenty-four hours following transfection, cellswere grown under normoxia (i.e., 20% oxygen) or hypoxia (i.e., 1%oxygen) for additional 24 hours, following which anti-HIF-1α antibodywas used in IP experiments. IP samples were further subjected to WesternBlot analysis using either anti-HIF-1α antibody or anti-Flag antibody.As is shown in FIGS. 4 a-b, the anti-HIF-1α antibody was capable ofprecipitating both Flag-HIF-1α and Flag-MSF-A proteins under normoxiaconditions, and to a lesser extent under hypoxia conditions.

The p300 co-activator is associated with HIF-1α and MSF-A proteins—Totest the hypothesis that proteins, which are known to interact with theHIF-1α protein, such as the co-activator p300, are associated with MSF-Aand HIF-1α, cells were co-transfected with the pcDNA3.1-HIF-1α and thep3xFlag-MSF-A expression vectors and IP experiments were performed usingeither anti-FLAG or anti-HIF-1α antibodies. As shown in FIGS. 5 a-c,while the anti-HIF-1α antibody was capable of precipitating the p300protein in both cells co-transfected with the pcDNA3.1-HIF-1α and thep3xFlag-cmv-25 expression vector alone and cells co-transfected with thepcDNA3.1-HIF-1α and the p3xFlag-MSF-A vectors, the anti-Flag antibodywas capable of precipitating the p300 protein only in cells that wereco-transfected with the pcDNA3.1-HIF-1α and the p3xFlag-MSF-A vectors.These results demonstrate that the MSF-A protein interacts with theHIF-1α protein complex.

Altogether, these results demonstrate that MSF-A strongly associateswith HIF-1α or HIF-1α complexes.

Example 2 MSF-A Upregulates HIF-1 Transcriptional Activity

To investigate the effect of MSF-A binding on HIF-1α function, thetranscriptional activity of the HIF-1 complex was determined using areporter gene assay as previously described (Mabjeesh N J et al. 2ME2inhibits tumor growth and angiogenesis by disrupting microtubules anddysregulating HIF. Cancer Cell 2003; 3: 363-375).

Experimental Results

MSF-A overexpression upregulates HIF-1 transcriptional activity—Theeffect of MSF-A on HIF-1α transcriptional activity was determined inPC-3 cells which were transiently co-transfected with a reporter plasmidcontaining the luciferase gene under the control of hypoxia responseelement (HRE) from the VEGF promoter (Post, D. E. and Van Meir, E. G.Generation of bidirectional hypoxia/HIF-responsive expression vectors totarget gene expression to hypoxic cells. Gene Ther., 8: 1801-1807, 2001)and with either the p3xFlag-MSF-A vector or the expression vector alone(p3xFlag-cmv-25). Following transfection, cells were grown undernormoxia or exposed to hypoxia. In cells co-transfected with theluciferase reporter plasmid and the expression vector alone, hypoxiainduced luciferase activity by more than 15-fold as compared tonormoxia. However, in cells co-transfected with the luciferase reporterplasmid and the MSF-A vector (p3xFlag-MSF-A), hypoxia induced luciferaseactivity by 50-fold as compared to normoxia (FIG. 6). Similar resultswere obtained when HEK 293 cells were transfected with the same vectors(not shown).

These results demonstrate that MSF-A over-expression enhances HIF-1transcriptional activity on target genes containing the HRE sequence.Thus, MSF-A upregulates the endogenous transcriptional activity ofHIF-1α.

Example 3 Association of ΔN-MSF-A with HIF-1α Results in Inhibition ofHIF-1α Transcriptional Activity

Members of the evolutionarily conserved septin family of genes have awell-conserved GTP binding domain and possess a GTPase activity. SEPT9has been shown to have a complex genomic architecture, such that up to15 different isoforms are possible by the shuffling of five alternateN-termini and three alternate C-termini. The MSF-A protein exhibitssequence homology with other members of the MSF superfamily except forthe first 25 amino acids at the N-terminal part (Kartmann B, Roth D.Novel roles for mammalian septins: from vesicle trafficking tooncogenesis. J Cell Sci 2001; 114: 839-844).

To investigate the role of the unique N-terminal sequence of MSF-A orthe common GTP-binding domain of MSF-A in the association and theactivation of HIF-1α (i.e., transcriptional activation of other genes),constructs of the ΔN-MSF-A (lacking the N-terminus) and ΔG-MSF-A(lacking the GTP binding site) were prepared and transiently expressedin cells.

Experimental Results

The activation of HIF-1α by MSF-A is dependent on the intact N-terminusof MSF-A protein and does not require the GTP binding domain—As is shownin FIG. 7, while the expression of wild-type (WT) MSF-A induced HIF-1αactivity (as detected by the luciferase reporter gene), the expressionof increasing amounts of the ΔN mutant of MSF-A (p3xFlag-ΔN-MSF-A)induced a dose-dependent inhibition of HIF-1α transcriptional activity.

These results demonstrate the use of the ΔN-MSF-A polypeptide (SEQ IDNO:10), or a polynucleotide encoding same (e.g., a polynucleotide setforth by SEQ

On the other hand, the expression of the ΔG mutant of MSF-A had nosignificant effect on HIF-1α activity (FIG. 8). Further IP experimentsshowed that both the ΔN-MSF-A and ΔG-MSF-A protein products are capableof binding the HIF-1α protein (FIGS. 9 a-c).

Altogether, these results indicate that the protein-protein associationbetween MSF-A and HIF-1α is not sufficient to activate HIF-1αtranscriptional activity and that full activation of HIF-1α by MSF-Arequires both, the GTP binding site and the intact N-terminus of MSF-A.

Example 4 MSF-A Over-Expression Induces Cell Proliferation and ColonyFormation

To further understand the mechanisms involved in upregulation of HIF-1αactivity via the association with MSF-A and to understand the role ofMSF-A in cancer cells, PC-3 cells were stably transfected with MSF-A(using the p3xFLAG-MSF-A vector) and neomycin resistant clones wereobtained.

Experimental Results

MSF-A stable PC-3 transfectants express MSF-A to various extents andexhibit increase HIF-1α transcriptional activity—The expression level ofboth recombinant MSF-A and endogenous HIF-1α were analyzed using WesternBlot analyses. As is shown in FIG. 10 a, using the anti-Flag antibodyvarious extents of expression levels were observed in the differentMSF-A transfectants. In addition, the HRE-reporter gene assay showedthat MSF-A stably expressing cells exhibit increased HIF-1transcriptional activity (FIG. 10 b). As an internal control, cells wereco-transfected with both Rinella SV40-Luciferase and the fireflyHRE-Luciferase and were subjected to dual Luciferase assay. There wereno changes in Rinella SV40-Luciferase activity under hypoxia or betweenthe different clones compared to empty vector control or parental PC-3cells (data not shown).

MSF-A increases the expression of HIF-downstream genes—To avoid clonalvariation of MSF-A over-expressing cells, 30 neomycin resistant clonesfrom each expression vector were pooled [i.e., EV-transfected(designated PC-3 Neo) and MSF-A-transfected (PC-3-MSF-A)] and the pooledcells were tested for HIF-1 transcriptional activity. As is shown inFIG. 11, PC-3-MSF-A cells exhibited a significant increase in HIF-1transcriptional activity (tested by the luciferase assay) as comparedwith PC-3-Neo cells. To further demonstrate the effect of MSF-A on HIF-1target genes, semi-quantitative RT-PCR analyses were performed. Briefly,total RNA was prepared from both PC-3-Neo and PC-3-MSF-A pooled cellsand RT-PCR was employed using specific primers for VEGF (SEQ ID NOs:4200and 4201), HIF-1α (SEQ ID NOs:4202 and 4203), β-actin (SEQ ID NOs:4204and 4205), ET-1 (SEQ ID NOs:4206 and 4207), CA-IX (SEQ ID NOs:4208 and4209) and Glut1 (SEQ ID NOs:4210- and 4211) (FIGS. 12 a-f). These RT-PCRanalyses demonstrated that while the HIF-1α mRNA level was unchanged inboth PC-3-MSF-A and PC-3-Neo cells (FIG. 12 e), the mRNA levels of theangiogenic factor VEGF were significantly higher in PC-3-MSF-A cellsthan in PC-3-Neo cells (FIG. 12 a). Other HIF-1 target genes includingGlut1 (FIG. 12 b), CA-IX (FIG. 12 d) and ET-1 (FIG. 12 c) were alsoupregulated to various extents in PC-3-MSF-A cells.

MSF-A increases cell proliferation and colony formation—To test theeffect of MSF-A on cell proliferation, the XTT assay was employed onPC-3-MSF-A and PC-3-Neo cells. As is shown in FIG. 13, under bothnormoxia and hypoxia, the proliferation rate of PC-3-MSF-A cells washigher to a greater extent than the proliferation rate of PC-3-Neocells. In addition, when grown in soft agar, PC-3-MSF-A cells formedsignificantly more colonies, each exhibiting a larger size as comparedwith PC-3-Neo cells grown (FIGS. 14 a-g). Moreover, PC-3-MSF-A cellsalso showed higher plating efficiency (49.3%) as compared to PC-3-Neocells (16.8%) (p<0.001).

Altogether, these results demonstrate that stable over-expression ofMSF-A in PC-3 cells enhances proliferation and upregulates HIF-1 targetgenes in vitro.

Example 5 MSF-A Affects HIF-1α Stabilization

To elucidate the mechanism by which MSF-A involves with HIF-1 activity,the effects of MSF-A expression on HIF-1α protein stability wasexamined. As shown in FIG. 12 e, MSF-A expression does alter HIF-1α mRNAlevels. The present inventor hypothesized that MSF-A affects HIF-1αactivity by modulating HIF-1α post-transcriptional/translational events.To this end, PC-3 stably transfected cells were employed under normoxiaor hypoxia and the effect on HIF-1α ubiquitination was studied.

Experimental Results

MSF-A stabilizes HIF-1α protein by preventing its ubiquitination—Thehypoxic induction of HIF-1α was studied at shorter rate limiting timeperiods rather than at times enabling to reach steady state levels.After 4 hours of exposure to hypoxia, the levels of HIF-1α proteinexpressed in PC-3-MSF-A cells was significantly higher than in PC-3-Neocells (FIG. 15 a-b). As is shown in FIGS. 15 a-b, the differences inHIF-1α expression levels were dependent on the time of hypoxiainduction. While the level of HIF-1α was higher in PC-3-MSF-A cellsafter 4 hours of hypoxia, similar levels of HIF-1α were obtained inPC-3-Neo and PC-3-MSF-A cells after 8 hours of hypoxia induction (FIGS.15 a-b). Therefore, the effect of MSF-A on HIF-1α protein stability wasfurther examined using the protein translation inhibitor cycloheximide(CHX) (FIGS. 16 a-c) and a pulse-chase assay (FIGS. 17 a-b). In thepresence of CHX new protein synthesis is inhibited, thus HIF-1α levelswould predominantly reflect the degradation process of HIF-1α protein.PC-3-Neo and PC-3-MSF-A cells were exposed to CHX for various incubationperiods (between 0-45 minutes) and HIF-1α protein levels were analyzedby Western blot analysis and normalized to those of α-tubulin. Within 20minutes of exposure to CHX, HIF-1α protein levels from PC-3-Neo cellswere decreased to about 50% (FIGS. 16 a-c). Although the intensity ofthe HIF-1α signal is different at the zero time-point, the degradationrate of HIF-1α protein was faster in PC-3-Neo than in PC-3-MSF-A cells(FIGS. 16 a-c). This was further confirmed when cells were labeled with³⁵S-methionine and pulse-chased, after which HIF-1α protein levels wereanalyzed. The half-life of HIF-1α protein from PC-3-Neo cells was around25 minutes compared to 45 minutes in PC-3-MSF-A cells (FIGS. 17 a-b). Asis further shown by the slope of the two curves, the rates of HIF-1αprotein loss were slower in PC-3-MSF-A cells than in PC-3-Neo cells(FIG. 17 b).

The pattern of HIF-1α immunoreactive bands in different MSF-A stableclones was examined under hypoxia (where degradation does not takeplace). As is shown in FIGS. 18 a-b, the various clones exhibiteddifferences in the higher molecular weight bands of HIF-1α protein whichlikely reflect ubiquitinated- and polyubiquitinated-HIF-1α (Ub-HIF-1α)species. The pattern of HIF-1α ubiquitination was inversely correlatedwith the levels of MSF-A protein expression (FIG. 18 b). These resultsdemonstrate that over-expression of MSF-A reduced HIF-1α ubiquitinationspecies (i.e., the rate of degradation). These results suggest that theincrease transcriptional activity of HIF-1α which was induced byover-expression of MSF-A is likely to be a result of stabilization ofthe HIF-1α protein by the MSF-A protein.

To study the ubiquitination of the endogenous HIF-1α in cellsover-expressing MSF-A protein, the proteasome inhibitor MG-132 was used.Under these conditions HIF-1α is subjected to ubiquitination but can notbe degraded through the proteasome. As is shown in FIGS. 19 a-b,increasing doses of MG-132 induced the expression of HIF-1α andUb-HIF-1α in PC-3-Neo cells. On the other hand, the pattern of Ub-HIF-1αlevels in PC-3-MSF-A cells was less intense and exhibited lowermolecular weight species (FIG. 19 a). To confirm the ubiquitinationforms of HIF-1α, PC-3-Neo and PC-3-MSF-A were treated with MG-132 andsubjected to IP with HIF-1α antibody. Immunoprecipitates wereimmunoblotted in parallel with either HIF-1α (FIG. 20 a) or ubiquitin(FIG. 20 b) antibodies. Again, the levels of Ub-HIF-1α protein werelower in PC-3-MSF-A cells than in PC-3-Neo cells (FIG. 20 a).

Altogether, these results demonstrate that the activation of HIF-1 byMSF-A is mediated through HIF-1α protein stabilization. MSF-A proteininteracts with HIF-1α protein under normoxic conditions to modulate itsubiquitination and thus escaping proteasomal degradation.

Example 6 Preparation and Characterization of MSF-A Antibodies

To further understand MSF-A role in the MSF-A-HIF-1α complex, MSF-Aspecific antibodies were generated and employed in Western Blot andfluorescence immunohistochemistry, as follows.

Experimental Results

Characterization of an MSF-A antibody—Following immunization with theN-terminal MSF-A peptide, sera was drawn from the immunized rabbits andwas tested on whole cell extracts prepared from PC-3 cells which werestably transfected with either the expression vector alone or the MSF-Avector (p3xFLAG-MSF-A). As is shown in FIGS. 10 a-c, while the anti-Flagantibody recognized the typical 70-kDa band only in MSF-A transfectedcells (FIG. 21 a), the immune serum recognized a 70 kDa band in cellstransfected with the expression vector alone or in cells transfectedwith the MSF-A expression vector (FIG. 21 c). In addition, as is furthershown in FIG. 21 c, the immune serum recognized a slightly highermolecular weight band in MSF-A transfected cells, but not in cellstransfected with the expression vector alone. The higher molecularweight band represents the Flag-tagged MSF-A in MSF-A transfected cells.Noteworthy, the preimmune serum which was diluted to the same extent(i.e., 1:500), revealed no binding signal (FIG. 21 b).

Confirmation of MSF-A antibody specificity—To further characterize thenew anti-MSF-A antibody (i.e., the immune serum), anti-Flagimmunoprecipitates, which were prepared from cells transfected with thep3xFLAG-MSF-A vector, were subjected to immunoblotting using the newanti-MSF-A antibody. The immune serum (i.e., the new anti-MSF-Aantibody) was capable of recognizing the typically immunoprecipitatedFlag-MSF-A protein (FIGS. 22 a-d). Flag-MSF-A was not recognized byother antibodies tested (data not shown).

Thus, these results demonstrate the generation of a new anti-MSF-Aantibody, which is capable of specifically interacting with the 70-kDaMSF-A protein using Western Blot.

Example 7 MSF-A and HIF-1 Alpha Co-Localize at the Cell Nuclei

To determine the localization of MSF-A, biochemical fractionation andlaser scanning confocal microscopy (LSCM) were employed, as follows.

Experimental Results

MSF-A is expressed in the nuclear fraction of a variety of cancerouscell lines—To identify the cellular localization of MSF-A, cytosolic andnuclear extracts were prepared from CL-1 and PC-3 cells which were grownunder normoxia or hypoxia for overnight. The nuclear and cytosolicfractions of the cells were subjected to Western Blot analysis using theanti-HIF-1α antibody following by the anti-MSF-A antibody (i.e., theimmune serum). As is shown in FIG. 23 a, in both CL-1 and PC-3 cellsupon hypoxic exposure HIF-1α protein was localized and accumulated inthe nuclear fraction. On the other hand, MSF-A was more predominantlylocalized in the nuclear fraction without any significant change in itslevels after hypoxia (FIG. 23 b). A slight increase in the expressionlevel of MSF-A protein was noted following hypoxia (FIG. 23 b). On theother hand, there was no significant difference in the expression levelof α-tubulin between the nuclear and cytosolic fractions (FIG. 23 c).

To further confirm the nuclear localization of MSF-A, PC3 cells weregrown under normoxia or hypoxia for 24 hours, following which they weresubjected to MSF-A immunohistochemistry and laser scanning confocalmicroscopy (LSCM). As is shown in FIGS. 24 a-d, the MSF-A staining waspredominantly in the nucleus. Further confirmation of the nuclearlocalization of MSF-A was obtained using anti-MSF-A/anti-HIF-1α doubleimmunohistochemistry. As is shown in FIGS. 25 a-f, MSF-A and HIF-1αco-localize to the cell nucleus. HIF-1α was barely detectable undernormoxic conditions but accumulated in the nucleus after exposure tohypoxia (FIGS. 25 a-b, green staining) while MSF-A was detected in thenucleus under both conditions, normoxia and hypoxia (FIGS. 25 c-d, redstaining). Overlay of both staining showed co-localization of HIF-1αwith MSF-A in the nucleus (FIGS. 25 e-f). Nuclear localization of MSF-Awas further confirmed by double-labeling with DAPI (data not shown). Theresults are consistent with the predicted sequence analysis of MSF-Aprotein that contains a bipartite nuclear targeting sequence at aminoacids 2-18 as set forth in SEQ ID NO:3.

Thus, these results demonstrate that MSF-A is a nuclear protein whichassociates and co-localizes with HIF-1α.

Evidence for HIF-1α/MSF-A interaction in vivo—To further confirm thatthe association of MSF-A with HIF-1α also occurs in vivo, prostatecancer PC-3 and CL-1 cells, which express substantial levels of theHIF-1α protein under normoxic conditions were used in an IP-IBexperiment. PC-3 or CL-1 cells were grown under normoxia or hypoxia andwhole cell extracts were then subjected to co-immunoprecipitation usingthe anti-HIF-1α antibody. The immunoprecipitates were then subjected toWestern Blot analysis using either the anti-HIF-1α or the anti-MSF-Aantibody. As is shown in FIGS. 26 a-c, under normoxic conditions, theanti-HIF-1α antibody was capable of immunoprecipitating both HIF-1α andMSF-A proteins. Under hypoxia, although there was a higher amount ofHIF-1α protein within the immunoprecipitate its interaction with MSF-Aprotein was much weaker as demonstrated in two different cell lines.Thus, under hypoxic conditions, MSF-A dissociates from HIF-1α. Theseresults are in good agreement with the results observed with 293transfected cells (see FIGS. 3 a-f for comparison). FIG. 27 depicts onesuggested model for MSF-A and HIF-1α interaction under normoxia andhypoxia.

Altogether, these results demonstrate that while under normoxiaconditions MSF-A associates with HIF-1α, and under hypoxia conditionsMSF-A dissociates from HIF-1α. Thus, the interaction between endogenousHIF-1α and MSF-A protein is O₂-dependent.

Example 8 The Effects of MSF-A on HIF-1, Tumor Growth and AngiogenesisIn Vivo

The effects of MSF-A on HIF-1, tumor growth and angiogenesis in vivo. Toexamine the effect of MSF-A on tumor growth, subcutaneous xenograftmouse tumor models were induced by the PC-3-MSF-A and PC-3-Neo cells.

Experimental Results

In this xenograft model, tumors derived from PC-3-MSF-A cells appearedearlier and exhibited increased mean volume (about 2-fold) compared totumors derived from PC-3-Neo cells (FIG. 28). Although the difference intumor volume was not statistically significant, the mean weight ofPC-3-MSF-A tumors was significantly heavier than the wild-type tumors(FIG. 29). Most importantly, the phenotype of the tumors was strikinglydifferent. Macroscopic and histological examination showed thatPC-3-MSF-A tumors were more pleomorphic, aggressive and invasive, andthere were only scattered small areas of necrosis, whereas a large areaof central necrosis was observed in PC-3-Neo cell derived xenografts(not shown). It was also found that MSF-A over-expression significantlyincreased intratumoral cell proliferation and vascular density (FIGS. 30a-h). RT-PCR analysis of RNA derived from the tumors showed that theexpression level of selected HIF-target genes, including VEGF and CA-IX,were elevated in PC-3-MSF-A tumors compared to PC-3-Neo tumors (FIGS. 31a-f). As a control, it is shown that PC-3-MSF-A tumor cells stillexpress higher levels of MSF-A mRNA (FIG. 31 a). Collectively, the invitro and in vivo data indicate that MSF-A affects HIF-1 transcriptionalactivation, cell proliferation and tumor angiogenesis.

Example 9 MSF-A Expression in Common Human Tumors

Since under normal oxygen conditions HIF is induced by a number ofoncogenes (e.g., AKT, Src, Ras), and since MSF-A was found to upregulateHIF-1α expression in vitro, the present inventor has hypothesized thatMSF-A is involved in HIF-1α expression in cancerous cells. Toinvestigate whether MSF-A is expressed in cancerous cells, whole cellsextracts prepared from various cancerous cell lines were subjected toWestern Blot using the new anti-MSF-A antibody (i.e., the immune serum).

Experimental Results

To examine the expression level of MSF-A in common human cancers, anexpression array with matched tumor/normal cDNAs from 68 tumors andcorresponding normal tissues from individual patients was employed. Thearray was hybridized with a probe (SEQ ID NO:4214) which reacts with allvariants of SEPT9 including MSF-A. For normalization, the array wasre-probed with a β-actin control probe (Ambion, Austin, Tx, USA),quantified and analyzed for comparison between normal versus tumorexpression profile. As shown in FIG. 32 a, strikingly SEPT9 gene wassignificantly over-expressed in ovarian tumor samples compared to othersamples tested. Interestingly, a lesser degree of SEPT9 over-expressionwas observed among samples of other female reproductive system includingbreast and uterus (FIG. 32 a). On the other hand, there was almost nochange noticed in expression level samples from renal, prostate orgastrointestinal tract tumors (FIG. 32 a).

To specifically follow the expression level of MSF-A, the RT-PCRanalysis was extended in prostate cancer where it was originally foundto interact with HIF-1α. RNA samples from various prostate cell linesand xenografts exhibited higher expression level of MSF-A mRNA inprostate cancer samples compared to those of RNA derived from normalprostate tissue (FIGS. 32 b-d).

Analysis and Discussion

In the present study, a novel regulatory pathway was identified in whichMSF-A, a member of the mammalian septin gene family affectstumorigenesis through, at least in part, the activation of theHIF-dependent response system. These results show that MSF-A proteindirectly interacts with HIF-1α but not with HIF-1β. Overexpression ofMSF-A leads to activation of HIF-1 and upregulation HIF-downstreamgenes, in vitro and in vivo. Most importantly, the findings of thepresent study demonstrate that MSF-A promotes proliferation, tumorgrowth and vascularization.

HIF-1 is a master regulator of the hypoxic response pathway not only inphysiological processes but also in pathophysiological states such asischemia and cancer (Maxwell and Ratcliffe, 2002; Semenza, 2003; Wenger,2002). Apart from the relatively well-characterized mechanisms ofhypoxic HIF-1α subunit stabilization, many growth factors and cytokinesare known to stabilize HIF-1α under normoxic conditions. Despite thisgreat diversity, most of these growth factors might stabilize HIF-1α viacommon cellular kinase pathways including PI-3K and MAPK pathways,activated by cell type-specific receptors (Wenger, 2002). However, sofar it is not completely understood how HIF-1α is stabilized in cancercells under “normoxic” conditions. The importance and potentialtherapeutic benefits of the HIF pathway have driven the search for newregulatory components. To that end, new candidates which affect the HIFpathway were searched for. Co-immunoprecipitation experiments revealedan interaction between MSF-A and HIF-1α. MSF-A is a splice variant ofthe SEPT9 of the mammalian septin gene family (Macara et al., 2002) andwas first found as a fusion partner gene of MLL in a case oftherapy-related acute myeloid leukemia with a t(11,17)(q23;q25)translocation (Osaka et al., 1999; Taki, Ohnishi, Shinohara, Sako,Bessho, Yanagisawa and Hayashi, 1999). Septins were originallydiscovered in yeast and found to be involved in diverse cellularprocesses, including cytokinesis, vesicle trafficking, apoptosis andmaintenance of cell polarity (Hall and Russell, 2004). The family ofhuman septins shows considerable homology in the core GTP-bindingdomain, but divergence in the N and C terminals (Hall and Russell, 2004;Kartmann and Roth, 2001). While most of the available data on thebiology of septins are derived from yeast, little is known on thephysiological and the pathophysiological significance of septins inmammals. A number of evidences suggest a role of septins in oncogenesis.Some of septin genes (SEPT5, 6, 9 & 11) are involved in chromosomaltranslocations in myeloid leukemias with the formation of chimeric MLLfusion proteins (Hall and Russell, 2004). Second, it was found recentlythat SEPT9 is altered in ovarian cancer (Burrows, Chanduloy, Mcllhatton,Nagar, Yeates, Donaghy, Price, Godwin, Johnston and Russell, 2003)consistent with the results of the present study and that SEPT9 isamplified and over-expressed in breast cancer (Montagna, Lyu, Hunter,Lukes, Lowther, Reppert, Hissong, Weaver and Ried, 2003). Very recently,Scott et al. has reported a meticulous analysis of SEPT9 expression in awide range of human tumors (Scott et al., 2005). SEPT9 was found to beover-expressed in breast, CNS, endometrium, kidney, liver, lung,lymphoid, esophagus, ovary, pancreas, skin, soft tissue and thyroid(Scott et al., 2005). In this study MSF-A was found to be specificallyupregulated in prostate cancer cell lines and xenografts. Interestingly,HIF-1α overexpression is also observed in the majority of human cancers(Zhong, De Marzo, Laughner, Lim, Hilton, Zagzag, Buechler, Isaacs,Semenza and Simons, 1999). Since in most cancers the mechanisms of howHIF-1α is over-expressed are yet not known while VHL is not mutated,these data support the hypothesis that the interaction between these twocellular processes may have a role in tumor progression in certaincancers.

MSF-A expression augments the activity of HIF-1 and induces higherproliferation rates both, under normoxia and hypoxia as well as in vitroand in vivo. Furthermore, MSF-A affects the pattern of tumor necrosiswith overall increased vascularity within tumors. This is the firstobservation that a septin protein has effects on tumor angiogenesis.

A number of septins have been shown to bind and hydrolyze guaninenucleotide. However, the role of GTP binding and hydrolysis in septinfunction has not been fully elucidated (Field, al-Awar, Rosenblatt,Wong, Alberts and Mitchison, 1996; Gladfelter, Bose, Zyla, Bardes andLew, 2002; Mendoza, Hyman and Glotzer, 2002; Robertson, Church, Nagar,Price, Hall and Russell, 2004; Versele and Thomer, 2004). Deletion ofthe GTP binding domain in MSF-A, led to no change in HIF-1 activity.There was neither activation nor inhibition of HIF-1 function while themutant lacking the GTP binding site still has the ability to interactwith HIF-1α protein. On the other hand, deletion of the most variableN-terminal domain of MSF-A exhibited dominant negative effect on HIFtranscriptional activity and still has been bound to HIF-1α. Theseresults indicate that binding of MSF-A to HIF-1α is not sufficient toactivate the HIF complex but requires binding and/or hydrolysis of GTPupon MSF-A as well as intact N-terminal. It was shown previously thatRac1 of the small GTPase Rho family is activated in response to hypoxiaand is required for the induction of HIF-1α protein expression andtranscriptional activity in hypoxic cells (Hirota and Semenza, 2001).Very recently, Nagata and Inagaki have identified a Rho-guaninenucleotide exchange factor (GEF) as a binding partner for MSF-A,describing the first link between septins and Rho signaling (Nagata andInagaki, 2005). It is reasonable to speculate that the activation ofHIF-1 by Rho could be mediated through interactions involving MSF-A.

Mechanistically, these data show that MSF-A affects HIF-1α protein atthe posttranslational level through stabilizing the protein andpreventing its ubiquitination. Although the interaction between HIF-1αand MSF-A was predominantly under normoxia, the hypoxic induction ofHIF-1 was also increased with overexpression of MSF-A. It is not clearyet whether MSF-A inhibits HIF-1α ubiquitination by preventing itsproline hydroxylation or by modulating VHL E3-ligase activity. Furtherstudies are necessary to elucidate the exact mechanism by which MSF-Astabilizes HIF-1α.

The interactions between HIF-1α and MSF-A, in addition to theircolocalization, and the functional activation of HIF-1 by MSF-A mayrepresent the role of SEPT9 function in tumorigenesis.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification, to the same extentas if each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

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What is claimed is:
 1. A method of treating prostate cancer and/orinhibiting a growth of a prostate cancerous tumor and/or prostatecancerous metastases in an individual in need thereof comprisingproviding to the individual an MSF-A derived peptide or peptideanalogue, of up to 30 amino acid resides in length, which includes theamino acid sequence set forth in SEQ ID NO: 4213, thereby treating theprostate cancer and/or inhibiting the growth of the prostate canceroustumor and/or the prostate cancerous metastases in the individual.
 2. Themethod of claim 1, wherein said MSF-A derived peptide or peptideanalogue further comprises a modification of said peptide or peptideanalogue for increasing penetration into cells or increasing stabilitywhile in a body.
 3. The method of claim 2, wherein said modificationcomprises an N terminus modification, a C terminus modification, apeptide bond modification, a backbone modification or a residuemodification.